Small Molecule Ghrelin Receptor Inverse Agonists and Antagonists

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Small Molecule Ghrelin Receptor Inverse Agonists and Antagonists Kimberly O. Cameron,*,† Samit K. Bhattacharya,† and A. Katrina Loomis‡ †

Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, 610 Main Street, Cambridge, Massachusetts 02139, United States ‡ Pharmatherapeutics Precision Medicine, Pfizer Worldwide Research and Development, Eastern Point Road, Groton, Connecticut 06340, United States ABSTRACT: Ghrelin is an endogenous peptide hormone secreted primarily by the stomach and is involved in a number of physiological processes including growth hormone secretion, food intake, as well as energy and glucose homeostasis. The physiological actions of ghrelin are mediated through the growth hormone secretagogue receptor 1a (ghrelin receptor), a peptidic G-protein-coupled receptor. This target has attracted much interest, as agents that block ghrelin’s actions on its receptor are anticipated to be pharmaceutical interventions for a number of diseases. This review provides an overview of ghrelin biology with a focus on metabolic diseases and summarizes recent medicinal chemistry programs aimed at delivering small molecule ghrelin receptor antagonists and inverse agonists to the clinic.

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INTRODUCTION Ghrelin is the endogenous ligand for the peptidic G-proteincoupled receptor (GPCR) designated the growth hormone secretagogue receptor (ghrelin receptor, previously designated GRLN or GHS-R1a).1,2 The discovery of ghrelin and its receptor has an interesting history starting in the 1980s with the realization that synthetic opioid peptides had growth hormone releasing capabilities.3,4 The action of these peptides, coined growth hormone releasing peptides (GHRPs), was determined to be independent of the growth hormone releasing hormone (GHRH) receptor.5 This finding led to the pursuit of growth hormone secretagogues (GHSs) and their cognate receptor resulting in the discovery of the ghrelin receptor,6 along with the progression of potent GHSs to clinical trials for disorders related to GH deficiency.7−9 The ghrelin receptor was known as an orphan receptor until 1999 when its endogenous ligand, ghrelin, was purified from rat stomach extracts and later identified in human stomach extracts.1 Ghrelin is a 28 amino acid, neuroendocrine hormone secreted primarily by oxyntic glands in the gastric mucosa of the stomach and is also expressed in the hypothalamus, pituitary, pancreas, adipose tissue, as well as a number of other peripheral tissues. The wide distribution of ghrelin mRNA suggests its involvement in the modulation of a number of physiological functions. In addition to its well-documented effect on growth hormone secretion, ghrelin has also been implicated in the secretion of adrenocorticotropic hormone (ACTH), cortisol, prolactin, and epinephrine.10 Ghrelin plays an important role in the acute regulation of appetite and on the maintenance of energy homeostasis.11 Ghrelin has been linked to reward pathways impacting food intake12,13 and alcohol dependence.14−16 Additionally, ghrelin has been implicated in a © XXXX American Chemical Society

number of physiological effects including the regulation of gastrointestinal,17−19 cardiovascular,10,20 and immune function.21−24 Finally, ghrelin has been suggested to play a role in glucose homeostasis25−27 and bone physiology.28,29 This review will summarize a number of recent attempts toward identification of ghrelin receptor antagonists for clinical application with a focus on small molecule approaches for the treatment of metabolic diseases.

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GHRELIN BIOLOGY Ghrelin is generated by proteolytic cleavage of the 117 amino acid pre-proghrelin precursor encoded by the gene GHRL. Cleavage of the N-terminal 23 amino acid signal peptide provides the 94 amino acid proghrelin peptide which, after further cleavage, gives rise to ghrelin as well as obestatin. Obestatin, a peptide hormone originally isolated from rat stomach,30 does not bind to the ghrelin receptor and has controversial biological activities.31 The hydroxyl group on the serine-3 residue of proghrelin is acylated, typically with an octanoyl group, through a unique post-translational event catalyzed by ghrelin O-acyltransferase (GOAT).32,33 Interest in GOAT inhibitors as a pharmacological approach to effectively block formation of ghrelin has been recently reviewed.34,35 Studies have demonstrated that truncation of ghrelin to the first four to five N-terminal amino acids is minimally required to activate the receptor and acylation on Ser3 is required for activation.1,36 The unacylated form of ghrelin (UAG) does not bind to or activate the ghrelin receptor despite having higher circulating plasma concentrations as compared to ghrelin.37,38 Received: February 27, 2014

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causative role for the observed obese phenotype.60−64 With starvation, ghrelin levels increase in order to maintain homeostatic intake of calories, as observed in humans undergoing diet-induced weight loss65 and patients with anorexia nervosa.66 A number of studies demonstrate that both baseline ghrelin and the pulsatile pattern of ghrelin are inhibited in obese subjects following gastric bypass surgery.65,67−69 However, conflicting data can also be found arguing against ghrelin’s role in the improvement in metabolic parameters after bariatric surgery.70,71 Acute administration of exogenous ghrelin stimulates food intake in rodents50,72,73 and humans.48 In contrast, anti-ghrelin antibodies, spiegelmers, and the selective “knockdown” of the ghrelin receptor in the hypothalamus lead to suppression of food intake in preclinical models.52,74,75 Despite these data, mice with a ghrelin loss of function76−79 showed no significant effects on body weight as compared to wild type mice on a standard diet.77 Ghrelin knockout mice on high fat diets, however, demonstrated increased fat oxidation and protection from diet-induced obesity.78 Mice lacking the ghrelin receptor had lower body weight as compared to wild-type animals despite having similar food intake. The weight difference was deemed modest and did not reach significance until 16 weeks of age.77 Double knockout mice lacking both ghrelin and its receptor demonstrate an increase in energy expenditure and decrease in body weight with no change in food intake as compared to both wild-type and mice lacking either ghrelin or the ghrelin receptor.79 The authors speculate that the presence of yet to be identified factors involved in the ghrelin pathway may allow for this mild phenotype observed with these animals. Ghrelin has been implicated as playing a role in hedonic eating which involves the consumption of food for pleasure rather than to satisfy the body’s need for calories, termed homeostatic feeding. Ghrelin levels were higher in humans12,80 exposed to pleasurable food as compared to those exposed to nonpleasurable foods. Intravenous administration of ghrelin to humans followed by exposure to images of food led to an increased response in brain regions involved in reward pathways as measured by functional magnetic resonance imaging (fMRI).81 In addition, a number of rodent models have demonstrated ghrelin’s ability to increase nonhomeostatic food intake. Peripheral administration of ghrelin in mice leads to increased preference for saccharine solution over water, and this preference was reduced in ghrelin receptor knockout mice.82 Central as well as peripheral administration of ghrelin to rats increased their motivation to obtain sucrose, as measured by number of lever presses. Administration of the peptidic ghrelin receptor antagonist 1 (JMV 2959, Figure 1)83 abolished this effect.84 Given ghrelin’s apparent role in addictive behaviors such as overeating, there is recent interest in ghrelin’s role in alcohol dependence. Plasma concentrations of ghrelin are significantly increased in alcoholics85 and during early abstinence from alcohol in alcohol-dependent subjects.86,87 In rodents, ghrelin administration both centrally and peripherally leads to increased alcohol consumption whereas this effect is attenuated in knockout animals or rodents treated with 1.14,15,88,89 The data described above suggest that ghrelin receptor antagonists or inverse agonists may be beneficial in the treatment of obesity and other addictive behaviors. The need for central exposure of drugs that block ghrelin’s actions on food intake remains an outstanding question. Exogenous administration of ghrelin to patients90 as well as rodents91

There is emerging data that suggest UAG may have significant biological functions including beneficial effects on insulin secretion and lipolysis39,40 and appears to act as an endogenous antagonist of ghrelin.41 The ghrelin receptor (GHS-R1a) is one of two known splice variants derived from the GHSR gene, the other being GHSR1b. It has seven transmembrane domains, whereas GHS-R1b has five transmembrane domains; ghrelin does not bind or activate this receptor and the functional importance of GHSR1b is unknown.6 The ghrelin receptor is highly conserved across species. For example, the rat and human receptors are highly homologous, differing by two amino acids.1 Expression of the ghrelin receptor can be found in the brain and a number of peripheral tissues such as pituitary, pancreas, heart, vasculature, and adrenal cortex.42 The ghrelin receptor activates multiple signaling pathways through Gαq/Gαi/Gα12/13 protein and β-arrestin recruitment. Given its multiple signaling pathways, identification of ligands that bind to the ghrelin receptor with resulting G protein biased signaling is of interest.43 In cell systems, the ghrelin receptor exhibits high constitutive activity44 (∼50% activity independent of ligand), and therefore, some drug discovery programs have actively pursued inverse agonists of this receptor. Inverse agonists, unlike receptor antagonists that block the actions of the endogenous agonist at the receptor, are able to decrease basal signaling in the absence of ligand. The discovery of human genetic mutations linked to short stature and obesity that do not affect binding of the ghrelin ligand but abolish the constitutive activity of the receptor provides rationale for this approach.45,46 The broad distribution of both ghrelin and its receptor suggests the potential for ghrelin to elicit a broad range of biological activities. An overview of both preclinical and human genetic data is provided for only a subset of diseases that were chosen based on the focus of most drug discovery programs. In some cases, data are confounding, and this may be due to the lack of distinction between ghrelin (acylated form) and UAG as well as the technical challenges in accurately measuring ghrelin due to its inherent instability.37,47

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GHRELIN AND ITS ROLE IN FOOD INTAKE The endogenous hormone, ghrelin, is the only peripheral, orexigenic hormone identified to date.48,49 Plasma ghrelin concentrations increase before meals and decline after eating in both rodents50 and humans,11,49 suggesting that ghrelin acts as a feeding signal. Activation of ghrelin receptors expressed centrally as well as peripherally on the vagal nerve is linked to ghrelin’s food intake effects. Within the arcuate nucleus of the hypothalamus, ghrelin activates homeostatic feeding via orexigenic neuropeptide Y (NPY) and agouti-related protein (AGRP) neurons while inhibiting anorexigenic proopiomelanocortin (POMC) neurons.51−53 Other sites within the brain express the ghrelin receptor and include the dorsal vagal complex,54 the ventral tegmental area (VTA),55 and the hypothalamic paraventricular nucleus.56 The mesolimbic dopaminergic system in the VTA is implicated in ghrelin’s effects on hedonic and reward-related behaviors leading to excessive intake of calories and other addictive behaviors which will be summarized below.57 Circulating ghrelin levels decrease with obesity in humans58 and in rodents.59 Humans with Prader−Willi syndrome (PWS), a rare genetic disorder resulting in severe hyperphagia and obesity, have elevated plasma ghrelin levels and indicate a B

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Figure 1. Peptidic ghrelin receptor antagonists.

dosing in healthy, older adults. Interestingly, autoradiography data in rats suggest that 3 has limited brain exposure, suggesting these effects are driven by a peripheral mechanism.105 In contrast, deletion of ghrelin in ob/ob mice led to improvements in glucose homeostasis and increases in insulin secretion.106 Deletion of ghrelin in mice also improved glucose tolerance and insulin sensitivity.107 Peripheral administration of the peptidic ghrelin receptor antagonist 2 ([D-Lys3]-GHRP-6, Figure 1)1 in ob/ob mice led to a decrease in body weight gain and improved glycemic control.108,109 To confirm that ghrelin has a direct effect on the pancreas, changes in glucose stimulated insulin release from the perfused rat pancreas were explored. Administration of ghrelin blocked insulin release in this system, whereas administration of antagonist, 2, led to significant enhancement of insulin release.96 Mechanistic studies attribute ghrelin’s effects in the pancreas to inhibition of glucose-stimulated cAMP production.110 As a whole, these data support the pursuit of pharmacological agents that selectively block ghrelin’s signaling through the ghrelin receptor as a novel mechanism to treat T2DM. Infusion of UAG in obese diabetics leads to improved glycemic control with concomitant decreases in ghrelin,39 suggesting an alternative approach where UAG or analogues of UAG could oppose ghrelin’s effects.41

that have undergone vagotomy does not demonstrate increases in food intake or weight gain, suggesting the importance of ghrelin receptors expressed on the vagus to the orexigenic effects of ghrelin. Another study, however, using alternative surgical vagotomy methods in rodents failed to inhibit feeding in response to peripheral ghrelin administration.92 Given these data, it is not clear if brain-penetrating ghrelin receptor antagonists are necessary for weight loss therapy, and this will require investigation.

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GHRELIN AND ITS ROLE IN GLUCOSE HOMEOSTASIS Type 2 diabetes mellitus (T2DM) is generally characterized by insulin resistance leading to hypersecretion of insulin and eventual pancreatic β-cell dysfunction. Recent literature suggests that ghrelin plays an important role in glucose homeostasis.93,94 Ghrelin, the ghrelin receptor, and GOAT are expressed in different cell types within the pancreas, suggesting a role in modulation of insulin secretion.95 Ghrelin appears to be released from the pancreas as determined by the significantly higher plasma concentrations of both ghrelin and UAG in the pancreatic vein compared to that of the artery.96 Endogenous levels of ghrelin are elevated in obese type 2 diabetics97 and these levels have an inverse correlation with insulin sensitivity in patients with metabolic syndrome. This is in contrast to UAG and total ghrelin levels, where plasma levels are negatively correlated to insulin sensitivity.98 Acute, exogenous administration of ghrelin decreases glucose-stimulated insulin secretion in both normal, healthy humans and rodents.26,99−102 In addition, infusion of ghrelin leads to a reduction in insulinstimulated glucose disposal in healthy and hypopituitary men, demonstrating that ghrelin’s effects are not due to changes in GH and cortisol.103 It was also reported, however, that administration of ghrelin to healthy volunteers at more physiological levels resulted in decreased insulin secretion with no effect on insulin sensitivity.100 Clinical studies with the ghrelin receptor agonists 3 (capromorelin, CP-424391)7 and 4 (ibutamoren, MK-677) (Figure 2)104 demonstrated increases in fasting glucose, HbA1c, and insulin resistance after chronic

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HUMAN GENETICS OF GHRELIN SIGNALING GENES On the basis of the body of literature indicating that ghrelin signaling plays a role in food intake and metabolic disease, ghrelin (GHRL), its receptor (GHSR), and activating enzyme GOAT (MBOAT4) have been the subject of numerous genetic association studies. Human genetic association studies can be used as a means to develop confidence in rationale for targeting a specific protein or pathway to achieve the desired therapeutic outcome or to identify potential safety concerns. Genetic association studies typically investigate whether the frequency of a genetic variant significantly differs based on disease state, phenotype, or biomarker. These studies can be hypothesis driven, as in candidate gene association studies that interrogate variants in specific genes, or hypothesis-free, as in genome wide association studies (GWAS) that interrogate variants across the genome. Numerous candidate gene association studies have been undertaken to explore the contribution of genetic variants in and near GHSR, GHRL, and GOAT with obesity related traits, stature, metabolic and cardiovascular traits, and reward mechanisms (reviewed in refs 111−113). The results of these studies have been generally inconsistent, failing to provide compelling genetic associations with most traits. In addition,

Figure 2. Small molecule ghrelin receptor agonists. C

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Metabolic disease is a complex phenotype influenced by genetic, epigenetic, and environmental factors. The genetic association data currently available do not provide definitive evidence that common variation in ghrelin signaling genes is associated with metabolic disease or related traits. The contribution of common variation in a single gene to complex disease etiology is likely to be small, and current genetic association studies may not be powered to detect the impact of genetic variation in ghrelin signaling genes with small effect sizes on metabolic phenotypes. While genetic data do not currently support a link between ghrelin signaling genes and metabolic disease, the limitations associated with genetic association studies preclude us from confirming that ghrelin is a poor therapeutic target for metabolic disease. In fact, there are examples, such as dipeptidyl peptidase-4 (DPP4), where current genetic data fail to predict a compelling therapeutic target for type 2 diabetes, but successful use of DPP4 inhibitors for T2DM suggests otherwise.116−119 Continued advances in technology and statistical analysis methodology in the field of human genetics will enable future studies to explore the impact of rare and low frequency variants and variants with very small effect sizes. These studies may provide additional insight into the relationship between ghrelin signaling and metabolic disease.

recent large scale GWAS results have been negative in these gene regions for obesity, metabolic, and cardiovascular related traits.114−121 Genetic association studies can provide valuable information linking a gene to a disease, endophenotype, or biomarker; however, lack of genetic association should be interpreted with caution. Genetic association studies focused on easily measurable cardiovascular and metabolic related traits are very well powered with some recent studies including over 100 000 subjects and more than 2 million variants across the genome. Despite the impressive improvements in genetic coverage, sample size, and analytical methods that have occurred over the past decade, there are still limitations that should be noted. Genome wide and candidate gene association studies do not cover all of the genetic variability present in a given gene, and there are limits to the effect sizes that can be detected. Thus, a lack of genetic association is rarely considered definitive. In addition, because regions of a chromosome are genetically linked, genetic associations will provide a link to a genetic locus but fine-mapping and functional follow-up studies are required to definitely determine the causal gene and variant in a given association locus. While several candidate gene association studies have been published supporting a role for ghrelin or its receptor in obesity, metabolic disease, and hypertension, the results have not been consistent.122−135 Recent large-scale genome wide association studies for BMI, T2DM, glycemia related traits, and blood pressure did not identify variants in or near ghrelin or its receptor that were significantly or suggestively associated with these traits.114−121 Candidate gene studies have investigated a link between variants in GHSR and left ventricular hypertrophy and coronary artery disease.136,137 The associations have not been replicated, and larger scale studies will be necessary to determine if an association exists. GWAS has thus far not identified a significant association between the GHSR gene region and cardiovascular disease.121,138 Small candidate gene studies have investigated the link between ghrelin and eating disorders as well as reward mechanisms in the form of amphetamine and alcohol dependency.139−142 Existing studies are inconclusive, and larger studies with increased statistical power will be necessary to confirm the existence of an association with these traits. No genome-wide association studies have identified a link between ghrelin and eating behavior or alcohol dependence; however, these studies have not been statistically well powered to detect small effects.143−146 Both genome wide association studies and candidate gene association studies support a role for GHSR genetic variation in stature.147,148 In addition, rare deleterious mutations in GHSR have been associated with short stature, further confirming the link between gene and phenotype.149 Given the link between the ghrelin receptor and stature, several studies have also looked for associations between stature and ghrelin genetic variants.139,148 To date, there is no compelling genetic evidence indicating that ghrelin is associated with stature, including a lack of association in a well-powered GWAS for height.113,147 Genetic variation in GOAT (MBOAT4) has been less well studied than ghrelin and its receptor. Only one small association study has been published that investigated a link between genetic variants in GOAT and anorexia nervosa; a nominally significant association was found.150 Genome wide association studies do not support a link between the GOAT gene region and any phenotypes studied to date.

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REVIEW OF SMALL MOLECULE GHRELIN RECEPTOR ANTAGONISTS/INVERSE AGONIST AGENTS

There have been a number of reports on ghrelin receptor antagonists/inverse agonists from various pharmaceutical companies (Figure 3).151,152 A recent survey of key databases for patent applications claiming compounds aimed at blocking ghrelin’s actions on its receptor is illustrated in Figure 4. With the identification of the endogenous ligand for the ghrelin receptor in 1999, patents began to appear in 2001 with numbers peaking in 2005. Despite a gradual decline in the number of published patent applications, interest in this target continues. As discussed previously, the ghrelin receptor has high constitutive activity, and therefore, an inverse agonist might be expected to provide additional in vivo efficacy over an antagonist; however, this has yet to be demonstrated physiologically based on compounds published to date. The reported compounds are generally characterized by high molecular weight and/or high lipophilicity which reflect typical physical property space for ligands of peptidergic targets.153,154 Many of the reported compounds are expected to have high Pgp liability and hence limited brain exposure as evidenced by the calculated efflux ratios in Figure 3 (cMDR1 BA/AB efflux ratio of >2.5 is predictive of reduced brain exposure).155,156 To our knowledge only one compound to date has advanced to clinical trials. Past reviews151,152 have described efforts from a number of contributors to the field and examples of compounds delivered from Abbott (5, 6, and 7),157−159 Bayer (8 (YIL-780) and 9 (YIL-870)),160 Aeterna Zentaris (10 (JMV 3002)),161 and Merck (11)162 can be found in Figure 3. The current review will summarize publications related to small molecule ghrelin receptor antagonists/inverse agonists that have been publicly disclosed from 2009 through the end of 2013. D

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Figure 3. Recent examples of ghrelin receptor antagonists/inverse agonists. Footnotes for the table are the following: aCalculated log D (ACD Labs program, version 12, pH 7.4). bTopological polar surface area.163 cCalculated MDR1 BA/AB ratio useful for indications where CNS penetration is desired.156

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indication.162 While a number of compounds were specifically claimed and in vivo food intake and body weight studies described, no specific data for any compound were provided. In 2009, Merck disclosed their efforts in targeting a ghrelin

MERCK

Merck had earlier published on a pyrazole series of ghrelin receptor antagonists (e.g., 11) intended for an antiobesity E

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bioavailability in rat, it was dosed iv and had a brain/plasma ratio of 0.15. Compound 12 was progressed to food intake studies in lean as well as in diet-induced obese (DIO) mice but did not exhibit any efficacy which the authors suggested was due to inadequate brain exposure. Compound 12, when dosed in a fasting induced refeeding lean rat model, demonstrated a 52% reduction in food intake. However, adverse effects were observed at a higher dose, leading the authors to suspect that the decreases in food intake may be due to off-target effects. Optimization for increased bioavailability and brain exposure by replacing the urea moiety with heterocycles led to analogues that displayed agonist activity. The authors report that they suspended efforts in this series in favor of an alternative series. No development update is available for either of the two disclosed series.

receptor antagonist/inverse agonist as an antiobesity agent in a different series.165 Merck’s starting point was a screening hit,

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ELIXIR Elixir reported on a series of sulfonamides as ghrelin receptor antagonists that were targeted for both obesity and diabetes indications.166,167 There is no report on the origin of these compounds. Compound 13 (Figure 6) exhibited nanomolar

Figure 4. Number of published patents focused on blocking ghrelin’s actions on its receptor by year. A combination of keywords and index terms were used to retrieve patent data from Thomson Reuters Cortellis, Thomson Reuters Intergity, and SciFinder.164

12a (Figure 5), which, in a competitive binding assay using labeled 4 (IC50 = 13 nM) as the radioligand, demonstrated potent binding to the ghrelin receptor. In addition, 12a demonstrated antagonism in an aequorin bioluminescence functional assay in HEK-293 cells expressing human ghrelin receptor (Ca2+ IC50 = 40 nM). The screening hit had high MW, poor pharmacokinetic (PK) properties, and off-target β3 adrenergic activity. The medicinal chemistry optimization goals were to eliminate the off-target activity, reduce MW, and improve ADME properties. The authors showed that while the basic amine was important for ghrelin receptor activity, the N-substituent was not important and allowed for removal of the amino-3-(aryloxy)propan-2-ol group, a known β3 adrenergic pharmacophore. Thus, the screening hit could be truncated to provide 12b. The authors replaced the hexyl chain on the lefthand portion of the molecule with a number of alternatives and found an increase in potency when replacing the hexyl group with phenyl or benzyl moieties. Eventually a naphthylethyl moiety (12) was identified as the preferred motif, with the individual enantiomers showing no difference in binding potency. Compound 12 was a potent human ghrelin receptor antagonist in the aqueorin functional assay (IC50 = 11 nM), an inverse agonist in an inositol phosphate accumulation scintillation proximity assay (SPA) in HEK-293 cells expressing rat ghrelin receptor (EC50 = 1 nM), and did not bind to the β3 adrenergic receptor. Compound 12 also inhibited ghrelin stimulated GH release in rat pituitary cells. While 12 had poor

Figure 6. Elixir ghrelin receptor antagonist. Ki is from a human ghrelin receptor binding assay.

binding affinity and inhibited ghrelin-stimulated activation in a luminescence-based reporter assay. Compound 13 showed good oral bioavailability (63%) in rats. Compounds in the sulfonamide series blocked ghrelin-induced as well as spontaneous food intake in normal mice. In mice on a high fat diet (HFD), oral treatment of 13 for 7 days showed a dose dependent decrease in body weight. In this 7-day study, no improvements in glucose disposal were observed but reductions in the amount of insulin required to dispose the glucose were observed. Compound 13 was dosed orally for 56 days and demonstrated sustained weight loss as well as greater efficiencies in glucose disposal with a substantial reduction in insulin response. In addition, quite remarkably, a 50% reduction in fat content was seen in the liver of the compound treated animals. Although no brain exposure data are reported, this compound would not be expected to have significant brain exposure given its high calculated MDR BA/AB ratio (6.6). As of 2009, Elixir was reported to be partnering with Novartis on

Figure 5. Optimization of Merck screening hit 12a to inverse agonist 12. Potency data are from a human ghrelin receptor binding assay. F

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Figure 7. Amgen series 1. Potency data are from a human ghrelin receptor aqueorin luminescence assay.

Figure 8. Amgen series 2. Potency data are from a human ghrelin receptor aqueorin luminescence assay.

and metabolic stability with a plan to combine the improvements from each region. Thus, removal of the amide in the valine−amide sector provided an equipotent compound. Replacing the piperidine-N-benzyl with the exocyclic cyclopropylmethylamino group provided a potent analogue that had good metabolic stability. On the phenoxy front, the authors discovered that disubstituted small electron withdrawing groups enhanced potency and provided metabolic stability. The best modifications from the left- and right-hand portions of the molecules were applied onto the benzodiazepinone scaffold to afford 15b which maintained ghrelin receptor potency but still lacked rat metabolic stability. Converting the scaffold to a lactam-based core afforded a potent analogue that had good metabolic stability in rat and human S9 fractions. Compound 15 was shown to have low in vivo clearance in rat and an oral bioavailability of 39%. No further functional or in vivo efficacy studies were reported by the authors. The development status of both series is not available.

development of this class of compounds that act as novel insulin sensitizers.168 No further updates are available.

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AMGEN

Amgen has published two reports on ghrelin receptor antagonists. The first report started with a lead identified in a HTS using an assay that sought inhibitors of intracellular calcium release induced by ghrelin in Chinese hamster ovary (CHO) cells that stably expressed human ghrelin receptor.169 The HTS hit, 14a (Figure 7), was later shown to be a partial agonist in a human inositol phosphate (IP) accumulation assay. Thus, the authors set out to optimize for potency and reduce the observed partial agonism. The indole and the piperazine core were difficult to replace, and structure−activity relationship (SAR) efforts focused on the biphenyl region. Identification of the α-methylpiperazine core along with addition of ortho substituents on the terminal phenyl provided potent ghrelin receptor binders; however, partial agonism remained an issue. Exploration of heterocyclic replacements of the terminal phenyl group led to the discovery that 4-pyridyl was devoid of partial agonism. Addition of an ortho substituent to the proximal phenyl ring provided tool compound 9 that exhibited no agonism. Compound 14 did not produce any growth hormone (GH) secretion in an ex vivo GH release experiment conducted in isolated rat pituitary cells, while it antagonized the GH release from rat pituitary cells upon ghrelin stimulation. This compound had high metabolic stability across species, was an excellent substrate for P-gp (MDR efflux ratio of 14), and had impaired brain penetration in mice (brain/plasma AUC = 0.16). Compound 14 demonstrated brain exposure in MDR1a deficient mice, thus enabling efficacy assessment for central nervous system (CNS) driven effects. However, no in vivo studies were reported. The second disclosure from Amgen was also focused on the identification of ghrelin receptor antagonists for the treatment of obesity.170 A HTS of the Amgen compound deck led to 15a (Figure 8) which was shown in an aequorin functional assay to be a full antagonist. Compound 15a was metabolically labile; thus, the authors set out to improve metabolic stability in this chemical space. The authors divided the HTS hit into four sectors and optimized each independently to improve potency

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GSK

GSK reported on their ghrelin receptor antagonist drug discovery program that commenced with a HTS hit, benzamide 16a (Figure 9), that was subsequently optimized to hydrazide 16b.171−173 The R chiral center at the benzylic carbon (assigned by vibrational circular dichroism) was found to be important for activity. Compound 16b had limited brain availability in rats (0.2% unbound fraction, brain tissue) and was able to inhibit ghrelin-induced GH release after intravenous administration. Since the piperazine N-des-methyl analogue was observed to be a major circulating metabolite in rat and dog, the authors chose to block the N-demethylation pathway by introducing a fused bicyclopiperazine ring. Preparation and testing of each diastereomer demonstrated that the R-C8, R-C10 diastereomer (as in compound 16 (GSK-1614343)) was preferred for antagonist activity as measured in a ghrelin stimulated intracellular calcium mobilization assay. To reduce overall lipophilicity of the analogues, the authors explored replacement of the benzyl aromatic ring to a 3-pyridine which helped maintain in vitro potency at a lower lipophilicity cost. Compound 16 showed good oral bioavailability in rats with low clearance and moderate half-life and similar brain G

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Figure 9. GSK series. Functional activity data are from a human ghrelin receptor FLIPR assay.

penetration as 16b (based on total brain to blood AUC ratio, free fractions not provided). It displayed antagonist activity in a FLIPR and a GTP-γ binding assay. When tested for inverse agonism in an inositol phosphate-SPA accumulation assay, compound 16 demonstrated no inverse agonism in contrast to the control compound, substance-P (a reported potent ghrelin receptor inverse agonist).174,175 Similar to its predecessor (16b), iv administration of compound 16 inhibited ghrelin induced GH-release in rats. Surprisingly, compound 16 displayed increases in food intake in rats when dosed orally or intravenously. Compound 16, either after 24 h or 7 day oral treatment, demonstrated statistically significant increases in food intake and body weight in WT mice but not in ghrelin receptor null mice, suggesting the food intake effects were ghrelin receptor mediated. In male Beagle dogs, compound 16 did not show any statistically significant change in food intake or body weight after 1 day of treatment. However, a 10-day oral treatment of compound 16 led to statistically significant increases in food intake and weight gain. Total plasma ghrelin levels were decreased after both acute and subchronic treatment in dogs. The differing results of a ghrelin receptor antagonist that is capable of inhibiting ghrelin-induced GH release and yet increasing food intake and body weight are attributed by the authors to a hitherto unknown mechanism (off-target activity) along with a potential effect of the compound on ghrelin receptor subtypes (GHS-R1b and yet uncloned).

A recent patent application from Tranzyme reported functional antagonism for the claimed compounds in an aequorin functional assay and inverse agonism based on an IP-one homogeneous time-resolved fluorescence (HTRF) assay.178,179 Given the novel nature of the macrocyclic analogues, the inventors shared a large amount of ADME data. For instance, compound 17 is reported to be a potent inverse agonist and has moderate stability in human liver microsomes (HLM). Compound 17 has low clearance in rodent PK studies and in Caco-2 cells has low permeability and high efflux (Papp(A→B) = 0.27; BA/AB ratio of 241). Despite the low permeability and high efflux numbers, the compound demonstrates an oral bioavailability of 11% in rodents. Compound 17, upon oral administration in an obese mouse model for 14 days, demonstrated a significant decrease in cumulative food intake. In addition, decreases in blood glucose, insulin, glucagon, and free fatty acids were achieved in an oral glucose tolerance test. No further updates are available on these leads.

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PROSIDION (OSI/ASTELLAS) Prosidion has published on orally bioavailable azaquinolines as ghrelin receptor antagonists that have decreased hERG offtarget activity relative to compound 8 (Bayer), the starting point for this series.180 Prosidion has also patented a series of analogues (e.g., 18, Figure 11) for both diabetes and obesity

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TRANZYME Tranzyme (now merged with Ocera Therapeutics) published on a novel class of macrocycles as ghrelin receptor modulators. One of the lead ghrelin agonist macrocycles is currently in clinic trials.176 Tranzyme has extended the field of macrocyclic ghrelin modulators to include ghrelin receptor antagonists/ inverse agonists. (e.g., 17, Figure 10).176,177

Figure 11. Prosidion representative analogue. Ki is from a human ghrelin receptor binding assay.

indications that are truncated versions of 8.181 Ghrelin receptor GTPγS binding data as well as ghrelin receptor competition IC50 were reported for selected analogues. No in vivo activity was provided for analogues in this series.

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HELSINN Helsinn has patented on a series of ureas for a number of disease areas including diabetes and obesity.182 Ghrelin receptor antagonist/agonist activity and in vivo mouse food intake inhibition data were reported. Compound 19 (Figure 12) is a representative example that is a potent antagonist (IC50(FLIPR) = 38 nM) and upon IP dosing in male C57BL/ 6J mice at 30 mpk showed 70% inhibition of food intake at 2 h which was sustained to 24 h. Ghrelin antagonist and inverse

Figure 10. Tranzyme macrocyclic series. IC50 is from a human ghrelin receptor binding assay. H

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moderate metabolism but excellent ghrelin receptor binding and antagonism. It still had affinity for P-gp but had a lower efflux ratio (6.6). Compound 20 inhibited gastric emptying in the presence or absence of ghrelin stimulation. In a fasted refed mouse model, which leads to increases in ghrelin secretion, compound 20 showed marked reductions in food intake over 4−6 h after a 10 mg/kg ip dose. Peripheral administration of compound 20 (ip 10 mg/kg) reversed ghrelin-induced (icv, 10 μg) food intake in rats. Finally, a reduction in glucose excursion was observed with compound 20 when dosed orally at 30 mpk in an ip glucose tolerance test in mice. The observed reduction in the glucose AUC (32% compared to vehicle) was similar to the positive control, glimepiride. These encouraging results led the team to invest further in a lead optimization effort, but no further updates have been reported.

Figure 12. Helsinn representative analogue. IC50 is from a human ghrelin receptor FLIPR assay.

agonist programs are listed on the Helsinn Web site for the treatment of obesity and are at the preclinical research stage.

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SANOFI Sanofi reported on their efforts to discover a ghrelin receptor antagonist for the treatment of obesity as well as diabetes.183 A high throughput screen (the particular assay utilized was not described) of the Sanofi-Aventis compound collection provided an indolinone hit (exemplified by 20a, Figure 13). These indolinones were known vasopressin-1 receptor (V1R) antagonists. As the authors pointed out, analogues with the core indolinone motif were also known ghrelin receptor agonists from leads (20c) published by Sumitomo.184 Thus, the medicinal chemistry optimization of the HTS hit set out with a goal of erasing V1R activity, improving ghrelin receptor antagonism, and improving the ADME profile. Removal of the N-sulfonyl group led to ablation of V1R activity. Transforming the urea linker to an amide off the C3 position of the indolinone provided 20b, the (+)-enantiomer of which demonstrated activity in a luciferase reporter assay in a human ghrelin receptor CHO-CreLuc cell line. The corresponding (−)-enantiomer was inactive in this assay. SAR in this scaffold showed that placing chlorine at the C6-position off the indolinone was essential for activity. A 4,6-dichloro substituted indolinone along with a 3,4-dichloro substituted phenyl ring at the C3 position of the indolinone scaffold provided a potent ghrelin receptor antagonist. Unfortunately, this analogue was metabolically labile with the major site of metabolism being the N-methylpiperazine. Replacements of the piperazine per se were not tolerated except for the more basic piperidine which had a high P-gp efflux ratio and hence would have decreased brain penetration. The authors therefore settled on the ethyl substituted piperazine (20). The (+)-enantiomer of 20 showed

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ASTRAZENECA AstraZeneca (AZ) recently disclosed a brain-penetrant ghrelin receptor antagonist/inverse agonist series intended for the treatment of obesity.185 Given the constitutive activity of the ghrelin receptor, AZ was interested in an inverse agonist for more robust efficacy. The authors utilized a brain-penetrant quinazolinone scaffold, exemplified by 9 and 21a (Figure 14), that had been previously published186 and used scaffoldhopping to identify a more lipophilically efficient pyrazolopyrimidinone core.154,187 Analogues from the pyrazolopyrimidinone were less lipophilic (via HPLC log D determination) as compared to the quinazoline and an alternative thienopyrimidinone core. In order to further reduce lipophilicity and minimize hERG activity, the C2-substituent was replaced by a pyridyl group that provided an improved hERG window. Thus, 21b was identified that had good solubility and no drug−drug interactions and maintained good ghrelin receptor binding potency. However, in a functional assay it exhibited undesired full agonism. The authors identified the piperidine nitrogen substituent as a vector for modulation of functional activity. Analogues with cyclic substituents off the piperidine-N showed reduced agonism and varied functional responses. Thus, the (R)-tetrahydrofuran analogue, 21, demonstrated inverse agonism while the corresponding (S)tetrahydrofuran was a neutral antagonist. Replacement of the tetrahydrofuran of compound 21 with cyclopentane provided

Figure 13. Sanofi ghrelin receptor antagonist series and Sumitomo ghrelin agonist. Potency data are from a human ghrelin receptor binding assay. I

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Figure 14. AstraZeneca series. Potency data are from a human ghrelin receptor binding assay.

Figure 15. Pfizer series. Potency data are from a human ghrelin receptor binding assay.

15) that was attractive given its fractional sp3 content (number of sp3 hybridized carbons/total carbon count, Fsp3 = 0.41) and the two point orthogonal “synthetically enabled” nitrogen vectors. In addition, this series of analogues displayed consistent inverse agonism, a feature that was desired in lead optimization efforts given the high constitutive activity of the ghrelin receptor as well as to streamline the drug discovery effort by focusing on a series unencumbered by functional switching. In an effort to reduce lipophilicity and identify a more ligand efficient substrate, the HTS-hit was optimized and changes were made to each of the vectors to arrive at 22b (PF04628935).191 Although a potent inverse agonist, compound 22b had undesired off-target pharmacology for the muscarinic acetylcholine receptor (mAChR, M2). Molecular modeling based on potential ligand binding sites in ghrelin receptor and M2 indicated that changes in the azetidine region of 22b could impart selectivity. This was achieved by replicating the conformational restriction provided by the ortho chlorine with an indane moiety as in 22 which concurrently led to the installation of a chiral center at the C5 position. The C5-R stereochemistry was shown to be preferred for potency and also imparted ghrelin receptor selectivity over M2. Combining the phenylacetamide group off the piperidine-N with the pyrimidinyl group on the indane provided analogue 22. This analogue was shown to have adequate brain exposure based on rat in vivo receptor occupancy. However, because of lack of a CNS target biomarker, Pfizer terminated the CNS approach. Pfizer redirected its research efforts toward a diabetes indication

an inverse agonist, while the regiosomeric tetrahydrofuran showed partial agonism. The (R)-tetrahydrofuran analogue 21 was a highly lipophilic efficient compound (LipE = LLE = 6.3)154,187 and was confirmed to be brain penetrant. Compound 21 was advanced to a food intake study in ghrelin receptor null and WT mice. Food intake in WT mice fell by 68% (compared to no change in the KO mice) over the first 2 h time period which was linked to the inverse agonist profile driving the observed effect. Pharmacokinetic analysis showed similar free exposure levels in KO and WT mice with free brain exposure covering the ghrelin receptor binding affinity for the first 2 h after dosing. This was in marked contrast to the quinazoline neutral antagonist 9 from Bayer. In a chronic weight loss experiment, treatment with 9 led to ∼16% weight loss over 14 days in DIO mice in both ghrelin receptor KO and WT animals, thus alluding to the fact that the observed weight loss from compound 9 was not ghrelin receptor-mediated but likely due to off-target pharmacology. No indication of further development of 21 has been reported.

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PFIZER Pfizer has published on a series of ghrelin receptor inverse agonists for obesity as well as diabetes indications.188−190 A HTS of the corporate compound collection was carried out using a radioligand binding assay. The binding hits were triaged in a follow-up GTP-γ-S functional assay. Hits that demonstrated an inverse agonist profile were prioritized. The initial campaign led to a spiro-piperidinyl-azetidine lead 22a (Figure J

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Given ghrelin’s role as a pleiotropic hormone, the safety of the mechanism will need to be clinically demonstrated. Safety is of paramount concern for treatments for chronic diseases such as obesity and diabetes, and it is unclear if blocking ghrelin’s actions on its receptor, either direct or indirect, in tissues such as brain, bone, and vasculature will lead to benefit or concern. Despite these challenges, given the prospect for ghrelin receptor antagonists/inverse agonists to be a first-in-class treatment for key diseases with high unmet medical need, there remains a clear desire to deliver potent and selective compounds to the clinic. Ongoing research on related targets including UAG and its unidentified receptor, GOAT, and GHSR1b may provide orthogonal approaches for pharmaceutical intervention.

given the emerging research that suggested ghrelin receptor signaling in the pancreas and the potential for ghrelin receptor antagonists to improve β cell function.26 Since compound 22 was shown to have multiple off-target activities in CEREP profiling, a physicochemistry-based strategy was pursued to increase polarity of the analogues while maintaining the ghrelin receptor potency in order to reduce off-target promiscuity. The imidazothiazole acetamide group (as in 22b) provided a reliable polar addition on one end, while the 6-methyl-4-pyrimidine group provided the best balance of ghrelin receptor potency and selectivity leading to compound 23 (PF-05190457). Compound 23 had an improved off-target profile, moderate clearance, and good permeability. In line with the MDR efflux ratio of 7 (predictive of reduced brain exposure), compound 23 showed decreased brain levels after 14-day dosing in rats.155,192 Compound 23 was an inverse agonist as measured in a europium-GTP functional assay and increased insulin secretion above an 11.2 mM glucose control in a human whole islet assay. Compound 23 completed 1 month rat and dog toxicology studies and is the first ghrelin receptor antagonist/inverse agonist to progress to clinical trials.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 617-551-3234. E-mail: kimberly.o.cameron@ pfizer.com.

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Notes

CHALLENGES AND FUTURE PROSPECTS There is a plethora of literature data linking ghrelin and its receptor to a number of physiological processes suggesting that ghrelin receptor may be a drug target of interest for the treatment of a number of diseases including obesity and diabetes. Despite strong interest in therapeutic approaches designed to interfere with ghrelin signaling by a number of pharmaceutical companies for over 2 decades, there is only one report of a small molecule antagonist/inverse agonist progressing to the clinic. Conflicting preclinical data using less-than-optimal tool compounds in different preclinical models, questionable translatability of these models to the clinic, and either paucity or unconvincing human genetics data have led to some uncertainty in the value of pursuing ghrelin receptor antagonists or inverse agonists for the treatment of metabolic diseases. From a medicinal chemistry perspective, challenges associated with the identification of potent compounds for this peptidic GPCR that have the desired functional profile in the appropriate physiochemical property space for a safe, oral drug may contribute to the lack of clinical successes. Antagonists with potency for targets in this class are generally lipophilic with high molecular weight which can often lead to issues associated with off-target promiscuity and solubility limited absorption. Off-target activity may not only lead to safety issues but can also confound interpretation of efficacy studies. For example, studies that demonstrate decreases in preclinical food intake must be viewed with skepticism in the absence of data (e.g., studies in ghrelin knockout mice) that confirm that the compound’s efficacy is due to interaction with the target. In programs in pursuit of antagonists, it is imperative to identify the appropriate in vitro assays that can clearly distinguish them from partial agonists, given the demonstrated ability for functional switching with minor structural changes within a series. Given the high constitutive activity of this receptor, an inverse agonist may be required for maximal efficacy. In addition, the possible need for compound distribution to the brain for certain indications adds additional complexity and our summary of published antagonists suggests that this challenge remains. Therefore, there still exists the need for additional tool compounds to fully assess the potential of this mechanism.

The authors declare the following competing financial interest(s): Authors are Pfizer employees and have stock/ shares in Pfizer, Inc. Biographies Kimberly O. Cameron received her Ph.D. in Organic Chemistry from the Univeristy of Colorado, Boulder, under the direction of Professor Gary A. Molander working on Lewis acid catalyzed annulation reactions. She received her B.A. from Rutgers University, NJ, and M.S. in Organic Chemistry from the Florida Institute of Technology. In 1993, she joined the Cardiovascular and Metabolic Diseases group at Pfizer in Groton, CT, as a medicinal chemist. She continues to work in the area of metabolic diseases as a Research Fellow for Pfizer Worldwide Medicinal Chemisry, located in Cambridge, MA. Samit K. Bhattacharya received his B.Sc. (Hons.) and M.Sc. in Organic Chemistry from Jadavpur University, Calcutta, India. He then moved on to University of Pennsylvania, Philadelphia, PA, where he earned his Ph.D. under the tutelage of Professor Jeffrey Winkler working on the total synthesis of anticancer drug paclitaxel (Taxol). Thereafter he moved to Columbia University, NY, as a Postdoctoral Fellow with Professor Samuel Danishefsky, working on the total synthesis of anticancer agent eleutherobin and ganglioside GM-1. In 1999, he joined the Cancer group in Pfizer, Groton, CT, as a medicinal chemist. He is currently a Senior Principal Scientist in the Cardiovascular and Metabolic Diseases group, located in Cambridge, MA. A. Katrina Loomis received her B.S. in Biology from Tulane University, LA, and her M.A. from The University of Texas at Austin where she studied reproductive endocrinology and toxicology. After several years working in biotechnology in the area of cancer drug discovery and directed protein evolution, she joined the Oncology Biology research unit at Pfizer in 2006 as a molecular and cellular biologist. Since 2007 she has been part of the human genetics community at Pfizer, focusing her efforts on applying genetics, ’omics, and population biology to drug discovery and development. She is currently an Associate Director in the Precision Medicine group focused on new target identification and target validation for metabolic diseases. K

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Stimulation of the growth hormone (GH)-insulin-like growth factor I axis by daily oral administration of a GH secretogogue (MK-677) in healthy elderly subjects. J. Clin. Endocrinol. Metab. 1996, 81, 4249− 4257. (9) Svensson, J.; Monson, J. P.; Vetter, T.; Hansen, T. K.; Savine, R.; Kann, P.; Bex, M.; Reincke, M.; Hagen, C.; Beckers, A.; Ilondo, M. M.; Zdravkovic, M.; Bengtsson, B. A.; Korbonits, M.; NN703 Clinical Research Group.. Oral administration of the growth hormone secretagogue NN703 in adult patients with growth hormone deficiency. Clin. Endocrinol. (Oxford, U. K.). 2003, 58, 572−580. (10) Nagaya, N.; Kojima, M.; Uematsu, M.; Yamagishi, M.; Hosoda, H.; Oya, H.; Hayashi, Y.; Kangawa, K. Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. Am. J. Physiol.: Regul., Integr. Comp. Physiol. 2001, 280, R1483−R1487. (11) Tschop, M.; Wawarta, R.; Riepl, R. L.; Friedrich, S.; Bidlingmaier, M.; Landgraf, R.; Folwaczny, C. Post-prandial decrease of circulating human ghrelin levels. J. Endocrinol. Invest. 2001, 24, RC19−RC21. (12) Monteleone, P.; Scognamiglio, P.; Monteleone, A. M.; Perillo, D.; Canestrelli, B.; Maj, M. Gastroenteric hormone responses to hedonic eating in healthy humans. Psychoneuroendocrinology 2013, 38, 1435−1441. (13) Menzies, J. R.; Skibicka, K. P.; Leng, G.; Dickson, S. L. Ghrelin, reward and motivation. Endocr. Dev. 2013, 25, 101−111. (14) Suchankova, P.; Steensland, P.; Fredriksson, I.; Engel, J. A.; Jerlhag, E. Ghrelin receptor (GHS-R1A) antagonism suppresses both alcohol consumption and the alcohol deprivation effect in rats following long-term voluntary alcohol consumption. PLoS One 2013, 8, e71284. (15) Jerlhag, E.; Egecioglu, E.; Landgren, S.; Salome, N.; Heilig, M.; Moechars, D.; Datta, R.; Perrissoud, D.; Dickson, S. L.; Engel, J. A. Requirement of central ghrelin signaling for alcohol reward. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 11318−11323. (16) Landgren, S.; Engel, J. A.; Hyytia, P.; Zetterberg, H.; Blennow, K.; Jerlhag, E. Expression of the gene encoding the ghrelin receptor in rats selected for differential alcohol preference. Behav. Brain. Res. 2011, 221, 182−188. (17) Masuda, Y.; Tanaka, T.; Inomata, N.; Ohnuma, N.; Tanaka, S.; Itoh, Z.; Hosoda, H.; Kojima, M.; Kangawa, K. Ghrelin stimulates gastric acid secretion and motility in rats. Biochem. Biophys. Res. Commun. 2000, 276, 905−908. (18) Fujino, K.; Inui, A.; Asakawa, A.; Kihara, N.; Fujimura, M.; Fujimiya, M. Ghrelin induces fasted motor activity of the gastrointestinal tract in conscious fed rats. J. Physiol. 2003, 550, 227−240. (19) Levin, F.; Edholm, T.; Schmidt, P. T.; Gryback, P.; Jacobsson, H.; Degerblad, M.; Hoybye, C.; Holst, J. J.; Rehfeld, J. F.; Hellstrom, P. M.; Naslund, E. Ghrelin stimulates gastric emptying and hunger in normal-weight humans. J. Clin. Endocrinol. Metab. 2006, 91, 3296− 3302. (20) Tritos, N. A.; Kissinger, K. V.; Manning, W. J.; Danias, P. G. Association between ghrelin and cardiovascular indexes in healthy obese and lean men. Clin. Endocrinol. (Oxford, U. K.) 2004, 60, 60−66. (21) Dixit, V. D.; Taub, D. D. Ghrelin and immunity: a young player in an old field. Exp. Gerontol. 2005, 40, 900−910. (22) Dixit, V. D.; Schaffer, E. M.; Pyle, R. S.; Collins, G. D.; Sakthivel, S. K.; Palaniappan, R.; Lillard, J. W., Jr.; Taub, D. D. Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. J. Clin. Invest. 2004, 114, 57−66. (23) Dixit, V. D. Adipose-immune interactions during obesity and caloric restriction: reciprocal mechanisms regulating immunity and health span. J. Leukocyte Biol. 2008, 84, 882−892. (24) Baatar, D.; Patel, K.; Taub, D. D. The effects of ghrelin on inflammation and the immune system. Mol. Cell. Endocrinol. 2011, 340, 44−58. (25) Meyer, C. Final answer: Ghrelin can suppress insulin secretion in humans, but is it clinically relevant? Diabetes 2010, 59, 2726−2728. (26) Tong, J.; Prigeon, R. L.; Davis, H. W.; Bidlingmaier, M.; Kahn, S. E.; Cummings, D. E.; Tschop, M. H.; D’Alessio, D. Ghrelin

ACKNOWLEDGMENTS The authors acknowledge Philip Carpino and Margaret Jackson for their critical review of this article and Vineet Sardar for performing the patent literature search.

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ABBREVIATIONS USED ACTH, adrenocorticotropic hormone; AGRP, agouti-related protein; AUC, area under the curve; AZ, AstraZeneca; BMI, body mass index; BW, body weight; CNS, central nervous system; DIO, diet-induced obese; FI, food intake; fMRI, functional magnetic resonance imaging; GWAS, genome wide association studies; GOAT, ghrelin O-acyltransferase; GPCR, G-protein-coupled receptor; GH, growth hormone; GHRH, growth hormone releasing hormone; GHRP, growth hormone releasing peptide; GHS, growth hormone secretagogue; GHSR1a (ghrelin receptor) growth hormone secretagogue receptor 1a; GHS-R1b, growth hormone secretagogue receptor 1b; GTP, guanosine 5′-triphosphate; FLIPR, fluorescence imaging plate reader; HFD, high fat diet; HTS, high throughput screen; HLM, human liver microsomes; ICV, intracerebroventricular; ip, intraperitoneal; iv, intravenous; MDR, multidrug resistance; mAChR, muscarinic acetylcholine receptor; NPY, neuropeptide Y; P-gp, P-glycoprotein; PK, pharmacokinetic; POMC, proopiomelanocortin; PWS, Prader−Willi syndrome; SAR, structure−activity relationship; SER, serine; SPA, scintillation proximity assay; TPSA, total polar surface area; T2DM, type 2 diabetes mellitus; UAG, unacylated ghrelin; V1R, vasopression1 receptor; VTA, ventral tegmental area; WT, wild type

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REFERENCES

(1) Kojima, M.; Hosoda, H.; Date, Y.; Nakazato, M.; Matsuo, H.; Kangawa, K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999, 402, 656−660. (2) IUPHAR Database. http://www.iuphar-db.org/DATABASE/ ObjectDisplayForward?familyId=28&objectId=246&familyType= RECEPTOR. (3) Bowers, C. Y.; Momany, F.; Reynolds, G. A.; Chang, D.; Hong, A.; Chang, K. Structure−activity relationships of a synthetic pentapeptide that specifically releases growth hormone in vitro. Endocrinology 1980, 106, 663−667. (4) Codd, E. E.; Shu, A. Y.; Walker, R. F. Binding of a growth hormone releasing hexapeptide to specific hypothalamic and pituitary binding sites. Neuropharmacology 1989, 28, 1139−1144. (5) Cheng, K.; Chan, W. W.; Barreto, A., Jr.; Convey, E. M.; Smith, R. G. The synergistic effects of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 on growth hormone (GH)-releasing factor-stimulated GH release and intracellular adenosine 3′,5′-monophosphate accumulation in rat primary pituitary cell culture. Endocrinology 1989, 124, 2791−2798. (6) Howard, A. D.; Feighner, S. D.; Cully, D. F.; Arena, J. P.; Liberator, P. A.; Rosenblum, C. I.; Hamelin, M.; Hreniuk, D. L.; Palyha, O. C.; Anderson, J.; Paress, P. S.; Diaz, C.; Chou, M.; Liu, K. K.; McKee, K. K.; Pong, S. S.; Chaung, L. Y.; Elbrecht, A.; Dashkevicz, M.; Heavens, R.; Rigby, M.; Sirinathsinghji, D. J.; Dean, D. C.; Melillo, D. G.; Patchett, A. A.; Nargund, R.; Griffin, P. R.; DeMartino, J. A.; Gupta, S. K.; Schaeffer, J. M.; Smith, R. G.; Van der Ploeg, L. H. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996, 273, 974−977. (7) White, H. K.; Petrie, C. D.; Landschulz, W.; MacLean, D.; Taylor, A.; Lyles, K.; Wei, J. Y.; Hoffman, A. R.; Salvatori, R.; Ettinger, M. P.; Morey, M. C.; Blackman, M. R.; Merriam, G. R.; Capromorelin Study Group.. Effects of an oral growth hormone secretagogue in older adults. J. Clin. Endocrinol. Metab. 2009, 94, 1198−1206. (8) Chapman, I. M.; Bach, M. A.; Van Cauter, E.; Farmer, M.; Krupa, D.; Taylor, A. M.; Schilling, L. M.; Cole, K. Y.; Skiles, E. H.; Pezzoli, S. S.; Hartman, M. L.; Veldhuis, J. D.; Gormley, G. J.; Thorner, M. O. L

dx.doi.org/10.1021/jm5003183 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Perspective

identification of a potent inverse agonist. Mol. Endocrinol. 2003, 17, 2201−2210. (45) Pantel, J.; Legendre, M.; Cabrol, S.; Hilal, L.; Hajaji, Y.; Morisset, S.; Nivot, S.; Vie-Luton, M. P.; Grouselle, D.; de Kerdanet, M.; Kadiri, A.; Epelbaum, J.; Le Bouc, Y.; Amselem, S. Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature. J. Clin. Invest. 2006, 116, 760−768. (46) Holst, B.; Schwartz, T. W. Ghrelin receptor mutationstoo little height and too much hunger. J. Clin. Invest. 2006, 116, 637−641. (47) Blatnik, M.; Soderstrom, C. I. A practical guide for the stabilization of acylghrelin in human blood collections. Clin. Endocrinol. (Oxford, U. K.) 2011, 74, 325−331. (48) Wren, A. M.; Seal, L. J.; Cohen, M. A.; Brynes, A. E.; Frost, G. S.; Murphy, K. G.; Dhillo, W. S.; Ghatei, M. A.; Bloom, S. R. Ghrelin enhances appetite and increases food intake in humans. J. Clin. Endocrinol. Metab. 2001, 86, 5992−5995. (49) Cummings, D. E.; Purnell, J. Q.; Frayo, R. S.; Schmidova, K.; Wisse, B. E.; Weigle, D. S. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 2001, 50, 1714− 1719. (50) Tschop, M.; Smiley, D. L.; Heiman, M. L. Ghrelin induces adiposity in rodents. Nature 2000, 407, 908−913. (51) Kamegai, J.; Tamura, H.; Shimizu, T.; Ishii, S.; Sugihara, H.; Wakabayashi, I. Chronic central infusion of ghrelin increases hypothalamic neuropeptide Y and agouti-related protein mRNA levels and body weight in rats. Diabetes 2001, 50, 2438−2443. (52) Nakazato, M.; Murakami, N.; Date, Y.; Kojima, M.; Matsuo, H.; Kangawa, K.; Matsukura, S. A role for ghrelin in the central regulation of feeding. Nature 2001, 409, 194−198. (53) Elmquist, J. K.; Flier, J. S. Neuroscience. The fat−brain axis enters a new dimension. Science 2004, 304, 63−64. (54) Faulconbridge, L. F.; Cummings, D. E.; Kaplan, J. M.; Grill, H. J. Hyperphagic effects of brainstem ghrelin administration. Diabetes 2003, 52, 2260−2625. (55) Naleid, A. M.; Grace, M. K.; Cummings, D. E.; Levine, A. S. Ghrelin induces feeding in the mesolimbic reward pathway between the ventral tegmental area and the nucleus accumbens. Peptides 2005, 26, 2274−2279. (56) Olszewski, P. K.; Grace, M. K.; Billington, C. J.; Levine, A. S. Hypothalamic paraventricular injections of ghrelin: effect on feeding and c-Fos immunoreactivity. Peptides 2003, 24, 919−923. (57) Abizaid, A.; Liu, Z. W.; Andrews, Z. B.; Shanabrough, M.; Borok, E.; Elsworth, J. D.; Roth, R. H.; Sleeman, M. W.; Picciotto, M. R.; Tschop, M. H.; Gao, X. B.; Horvath, T. L. Ghrelin modulates the activity and synaptic input organization of midbrain dopamine neurons while promoting appetite. J. Clin. Invest. 2006, 116, 3229−3239. (58) Tschop, M.; Weyer, C.; Tataranni, P. A.; Devanarayan, V.; Ravussin, E.; Heiman, M. L. Circulating ghrelin levels are decreased in human obesity. Diabetes 2001, 50, 707−709. (59) Ariyasu, H.; Takaya, K.; Hosoda, H.; Iwakura, H.; Ebihara, K.; Mori, K.; Ogawa, Y.; Hosoda, K.; Akamizu, T.; Kojima, M.; Kangawa, K.; Nakao, K. Delayed short-term secretory regulation of ghrelin in obese animals: evidenced by a specific RIA for the active form of ghrelin. Endocrinology 2002, 143, 3341−3350. (60) Cummings, D. E.; Clement, K.; Purnell, J. Q.; Vaisse, C.; Foster, K. E.; Frayo, R. S.; Schwartz, M. W.; Basdevant, A.; Weigle, D. S. Elevated plasma ghrelin levels in Prader Willi syndrome. Nat. Med. 2002, 8, 643−644. (61) DelParigi, A.; Tschop, M.; Heiman, M. L.; Salbe, A. D.; Vozarova, B.; Sell, S. M.; Bunt, J. C.; Tataranni, P. A. High circulating ghrelin: a potential cause for hyperphagia and obesity in Prader−Willi syndrome. J. Clin. Endocrinol. Metab. 2002, 87, 5461−5464. (62) Haqq, A. M.; Farooqi, I. S.; O’Rahilly, S.; Stadler, D. D.; Rosenfeld, R. G.; Pratt, K. L.; LaFranchi, S. H.; Purnell, J. Q. Serum ghrelin levels are inversely correlated with body mass index, age, and insulin concentrations in normal children and are markedly increased in Prader−Willi syndrome. J. Clin. Endocrinol. Metab. 2003, 88, 174− 178.

suppresses glucose-stimulated insulin secretion and deteriorates glucose tolerance in healthy humans. Diabetes 2010, 59, 2145−2151. (27) Nass, R.; Gaylinn, B. D.; Thorner, M. O. The ghrelin axis in disease: potential therapeutic indications. Mol. Cell. Endocrinol. 2011, 340, 106−110. (28) Fukushima, N.; Hanada, R.; Teranishi, H.; Fukue, Y.; Tachibana, T.; Ishikawa, H.; Takeda, S.; Takeuchi, Y.; Fukumoto, S.; Kangawa, K.; Nagata, K.; Kojima, M. Ghrelin directly regulates bone formation. J. Bone Miner. Res. 2005, 20, 790−798. (29) Delhanty, P. J.; van der Eerden, B. C.; van Leeuwen, J. P. Ghrelin and bone. BioFactors 2013, 40, 41−48. (30) Zhang, J. V.; Ren, P. G.; Avsian-Kretchmer, O.; Luo, C. W.; Rauch, R.; Klein, C.; Hsueh, A. J. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science 2005, 310, 996−999. (31) Gargantini, E.; Grande, C.; Trovato, L.; Ghigo, E.; Granata, R. The role of obestatin in glucose and lipid metabolism. Horm. Metab. Res. 2013, 45, 1002−1008. (32) Yang, J.; Brown, M. S.; Liang, G.; Grishin, N. V.; Goldstein, J. L. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 2008, 132, 387−396. (33) Gutierrez, J. A.; Solenberg, P. J.; Perkins, D. R.; Willency, J. A.; Knierman, M. D.; Jin, Z.; Witcher, D. R.; Luo, S.; Onyia, J. E.; Hale, J. E. Ghrelin octanoylation mediated by an orphan lipid transferase. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6320−6325. (34) Mohan, H.; Unniappan, S. Discovery of ghrelin o-acyltransferase. Endocr. Dev. 2013, 25, 16−24. (35) Taylor, M. S.; Hwang, Y.; Hsiao, P. Y.; Boeke, J. D.; Cole, P. A. Ghrelin O-acyltransferase assays and inhibition. Methods Enzymol. 2012, 514, 205−228. (36) Bednarek, M. A.; Feighner, S. D.; Pong, S. S.; McKee, K. K.; Hreniuk, D. L.; Silva, M. V.; Warren, V. A.; Howard, A. D.; Van Der Ploeg, L. H.; Heck, J. V. Structure−function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J. Med. Chem. 2000, 43, 4370−4376. (37) Liu, J.; Prudom, C. E.; Nass, R.; Pezzoli, S. S.; Oliveri, M. C.; Johnson, M. L.; Veldhuis, P.; Gordon, D. A.; Howard, A. D.; Witcher, D. R.; Geysen, H. M.; Gaylinn, B. D.; Thorner, M. O. Novel ghrelin assays provide evidence for independent regulation of ghrelin acylation and secretion in healthy young men. J. Clin. Endocrinol. Metab. 2008, 93, 1980−1987. (38) Tong, J.; Dave, N.; Mugundu, G. M.; Davis, H. W.; Gaylinn, B. D.; Thorner, M. O.; Tschop, M. H.; D’Alessio, D.; Desai, P. B. The pharmacokinetics of acyl, des-acyl, and total ghrelin in healthy human subjects. Eur. J. Endocrinol. 2013, 168, 821−828. (39) Ozcan, B.; Neggers, S. J.; Miller, A. R.; Yang, H. C.; Lucaites, V.; Abribat, T.; Allas, S.; Huisman, M.; Visser, J. A.; Themmen, A. P.; Sijbrands, E.; Delhanty, P.; Van der Lely, A. J. Does des-acyl ghrelin improve glycemic control in obese diabetic subjects by decreasing acylated ghrelin levels? Eur. J. Endocrinol. 2014, 170, 799−807. (40) Benso, A.; St-Pierre, D. H.; Prodam, F.; Gramaglia, E.; Granata, R.; van der Lely, A. J.; Ghigo, E.; Broglio, F. Metabolic effects of overnight continuous infusion of unacylated ghrelin in humans. Eur. J. Endocrinol. 2012, 166, 911−916. (41) Delhanty, P. J.; Neggers, S. J.; van der Lely, A. J. Mechanisms in endocrinology: ghrelin: the differences between acyl- and des-acyl ghrelin. Eur. J. Endocrinol. 2012, 167, 601−608. (42) Davenport, A. P.; Bonner, T. I.; Foord, S. M.; Harmar, A. J.; Neubig, R. R.; Pin, J. P.; Spedding, M.; Kojima, M.; Kangawa, K. International Union of Pharmacology. LVI. Ghrelin receptor nomenclature, distribution, and function. Pharmacol. Rev. 2005, 57, 541−546. (43) Sivertsen, B.; Holliday, N.; Madsen, A. N.; Holst, B. Functionally biased signalling properties of 7TM receptors opportunities for drug development for the ghrelin receptor. Br. J. Pharmacol. 2013, 170, 1349−1362. (44) Holst, B.; Cygankiewicz, A.; Jensen, T. H.; Ankersen, M.; Schwartz, T. W. High constitutive signaling of the ghrelin receptor M

dx.doi.org/10.1021/jm5003183 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Perspective

(63) Feigerlova, E.; Diene, G.; Conte-Auriol, F.; Molinas, C.; Gennero, I.; Salles, J. P.; Arnaud, C.; Tauber, M. Hyperghrelinemia precedes obesity in Prader−Willi syndrome. J. Clin. Endocrinol. Metab. 2008, 93, 2800−2805. (64) Purtell, L.; Sze, L.; Loughnan, G.; Smith, E.; Herzog, H.; Sainsbury, A.; Steinbeck, K.; Campbell, L. V.; Viardot, A. In adults with Prader−Willi syndrome, elevated ghrelin levels are more consistent with hyperphagia than high PYY and GLP-1 levels. Neuropeptides 2011, 45, 301−307. (65) Cummings, D. E.; Weigle, D. S.; Frayo, R. S.; Breen, P. A.; Ma, M. K.; Dellinger, E. P.; Purnell, J. Q. Plasma ghrelin levels after dietinduced weight loss or gastric bypass surgery. N. Engl. J. Med. 2002, 346, 1623−1630. (66) Shiiya, T.; Nakazato, M.; Mizuta, M.; Date, Y.; Mondal, M. S.; Tanaka, M.; Nozoe, S.; Hosoda, H.; Kangawa, K.; Matsukura, S. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J. Clin. Endocrinol. Metab. 2002, 87, 240− 244. (67) Damjanovic, S. S.; Lalic, N. M.; Pesko, P. M.; Petakov, M. S.; Jotic, A.; Miljic, D.; Lalic, K. S.; Lukic, L.; Djurovic, M.; Djukic, V. B. Acute effects of ghrelin on insulin secretion and glucose disposal rate in gastrectomized patients. J. Clin. Endocrinol. Metab. 2006, 91, 2574− 2581. (68) Roth, C. L.; Reinehr, T.; Schernthaner, G. H.; Kopp, H. P.; Kriwanek, S.; Schernthaner, G. Ghrelin and obestatin levels in severely obese women before and after weight loss after Roux-en-Y gastric bypass surgery. Obes. Surg. 2009, 19, 29−35. (69) Samat, A.; Malin, S. K.; Huang, H.; Schauer, P. R.; Kirwan, J. P.; Kashyap, S. R. Ghrelin suppression is associated with weight loss and insulin action following gastric bypass surgery at 12 months in obese adults with type 2 diabetes. Diabetes Obes. Metab. 2013, 15, 963−966. (70) Beckman, L. M.; Beckman, T. R.; Earthman, C. P. Changes in gastrointestinal hormones and leptin after Roux-en-Y gastric bypass procedure: a review. J. Am. Diet. Assoc. 2010, 110, 571−584. (71) Stefater, M. A.; Wilson-Perez, H. E.; Chambers, A. P.; Sandoval, D. A.; Seeley, R. J. All bariatric surgeries are not created equal: insights from mechanistic comparisons. Endocr. Rev. 2012, 33, 595−622. (72) Wren, A. M.; Small, C. J.; Ward, H. L.; Murphy, K. G.; Dakin, C. L.; Taheri, S.; Kennedy, A. R.; Roberts, G. H.; Morgan, D. G.; Ghatei, M. A.; Bloom, S. R. The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology 2000, 141, 4325−4328. (73) Wren, A. M.; Small, C. J.; Abbott, C. R.; Dhillo, W. S.; Seal, L. J.; Cohen, M. A.; Batterham, R. L.; Taheri, S.; Stanley, S. A.; Ghatei, M. A.; Bloom, S. R. Ghrelin causes hyperphagia and obesity in rats. Diabetes 2001, 50, 2540−2547. (74) Shuto, Y.; Shibasaki, T.; Otagiri, A.; Kuriyama, H.; Ohata, H.; Tamura, H.; Kamegai, J.; Sugihara, H.; Oikawa, S.; Wakabayashi, I. Hypothalamic growth hormone secretagogue receptor regulates growth hormone secretion, feeding, and adiposity. J. Clin. Invest. 2002, 109, 1429−1436. (75) Becskei, C.; Bilik, K. U.; Klussmann, S.; Jarosch, F.; Lutz, T. A.; Riediger, T. The anti-ghrelin Spiegelmer NOX-B11-3 blocks ghrelinbut not fasting-induced neuronal activation in the hypothalamic arcuate nucleus. J. Neuroendocrinol. 2008, 20, 85−92. (76) Uchida, A.; Zigman, J. M.; Perello, M. Ghrelin and eating behavior: evidence and insights from genetically-modified mouse models. Front. Neurosci. 2013, 7, No. 121. (77) Sun, Y.; Wang, P.; Zheng, H.; Smith, R. G. Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4679−4684. (78) Wortley, K. E.; Anderson, K. D.; Garcia, K.; Murray, J. D.; Malinova, L.; Liu, R.; Moncrieffe, M.; Thabet, K.; Cox, H. J.; Yancopoulos, G. D.; Wiegand, S. J.; Sleeman, M. W. Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 8227−8232. (79) Pfluger, P. T.; Kirchner, H.; Gunnel, S.; Schrott, B.; Perez-Tilve, D.; Fu, S.; Benoit, S. C.; Horvath, T.; Joost, H. G.; Wortley, K. E.;

Sleeman, M. W.; Tschop, M. H. Simultaneous deletion of ghrelin and its receptor increases motor activity and energy expenditure. Am. J. Physiol.: Gastrointest. Liver Physiol. 2008, 294, G610−G618. (80) Monteleone, P.; Piscitelli, F.; Scognamiglio, P.; Monteleone, A. M.; Canestrelli, B.; Di Marzo, V.; Maj, M. Hedonic eating is associated with increased peripheral levels of ghrelin and the endocannabinoid 2arachidonoyl-glycerol in healthy humans: a pilot study. J. Clin. Endocrinol. Metab. 2012, 97, E917−E924. (81) Malik, S.; McGlone, F.; Bedrossian, D.; Dagher, A. Ghrelin modulates brain activity in areas that control appetitive behavior. Cell Metab. 2008, 7, 400−409. (82) Disse, E.; Bussier, A. L.; Veyrat-Durebex, C.; Deblon, N.; Pfluger, P. T.; Tschop, M. H.; Laville, M.; Rohner-Jeanrenaud, F. Peripheral ghrelin enhances sweet taste food consumption and preference, regardless of its caloric content. Physiol. Behav. 2010, 101, 277−281. (83) Moulin, A.; Brunel, L.; Boeglin, D.; Demange, L.; Ryan, J.; M’Kadmi, C.; Denoyelle, S.; Martinez, J.; Fehrentz, J. A. The 1,2,4triazole as a scaffold for the design of ghrelin receptor ligands: development of JMV 2959, a potent antagonist. Amino Acids 2013, 44, 301−314. (84) Skibicka, K. P.; Hansson, C.; Egecioglu, E.; Dickson, S. L. Role of ghrelin in food reward: impact of ghrelin on sucrose selfadministration and mesolimbic dopamine and acetylcholine receptor gene expression. Addict. Biol. 2012, 17, 95−107. (85) Kraus, T.; Schanze, A.; Groschl, M.; Bayerlein, K.; Hillemacher, T.; Reulbach, U.; Kornhuber, J.; Bleich, S. Ghrelin levels are increased in alcoholism. Alcohol.: Clin. Exp. Res. 2005, 29, 2154−2157. (86) Kim, D. J.; Yoon, S. J.; Choi, B.; Kim, T. S.; Woo, Y. S.; Kim, W.; Myrick, H.; Peterson, B. S.; Choi, Y. B.; Kim, Y. K.; Jeong, J. Increased fasting plasma ghrelin levels during alcohol abstinence. Alcohol Alcohol. 2005, 40, 76−79. (87) Koopmann, A.; von der Goltz, C.; Grosshans, M.; Dinter, C.; Vitale, M.; Wiedemann, K.; Kiefer, F. The association of the appetitive peptide acetylated ghrelin with alcohol craving in early abstinent alcohol dependent individuals. Psychoneuroendocrinology 2012, 37, 980−986. (88) Bahi, A.; Tolle, V.; Fehrentz, J. A.; Brunel, L.; Martinez, J.; Tomasetto, C. L.; Karam, S. M. Ghrelin knockout mice show decreased voluntary alcohol consumption and reduced ethanolinduced conditioned place preference. Peptides 2013, 43, 48−55. (89) Landgren, S.; Simms, J. A.; Hyytia, P.; Engel, J. A.; Bartlett, S. E.; Jerlhag, E. Ghrelin receptor (GHS-R1A) antagonism suppresses both operant alcohol self-administration and high alcohol consumption in rats. Addict. Biol. 2012, 17, 86−94. (90) le Roux, C. W.; Neary, N. M.; Halsey, T. J.; Small, C. J.; Martinez-Isla, A. M.; Ghatei, M. A.; Theodorou, N. A.; Bloom, S. R. Ghrelin does not stimulate food intake in patients with surgical procedures involving vagotomy. J. Clin. Endocrinol. Metab. 2005, 90, 4521−4524. (91) Date, Y.; Murakami, N.; Toshinai, K.; Matsukura, S.; Niijima, A.; Matsuo, H.; Kangawa, K.; Nakazato, M. The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats. Gastroenterology 2002, 123, 1120−1128. (92) Arnold, M.; Mura, A.; Langhans, W.; Geary, N. Gut vagal afferents are not necessary for the eating-stimulatory effect of intraperitoneally injected ghrelin in the rat. J. Neurosci. 2006, 26, 11052−11060. (93) Verhulst, P. J.; Depoortere, I. Ghrelin’s second life: from appetite stimulator to glucose regulator. World J. Gastroenterol. 2012, 18, 3183−3195. (94) Dezaki, K.; Yada, T. Islet beta-cell ghrelin signaling for inhibition of insulin secretion. Methods Enzymol. 2012, 514, 317−331. (95) An, W.; Li, Y.; Xu, G.; Zhao, J.; Xiang, X.; Ding, L.; Li, J.; Guan, Y.; Wang, X.; Tang, C.; Li, X.; Mulholland, M.; Zhang, W. Modulation of ghrelin O-acyltransferase expression in pancreatic islets. Cell. Physiol. Biochem. 2010, 26, 707−716. (96) Dezaki, K.; Sone, H.; Koizumi, M.; Nakata, M.; Kakei, M.; Nagai, H.; Hosoda, H.; Kangawa, K.; Yada, T. Blockade of pancreatic N

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Perspective

islet-derived ghrelin enhances insulin secretion to prevent high-fat dietinduced glucose intolerance. Diabetes 2006, 55, 3486−3493. (97) Rodriguez, A.; Gomez-Ambrosi, J.; Catalan, V.; Gil, M. J.; Becerril, S.; Sainz, N.; Silva, C.; Salvador, J.; Colina, I.; Fruhbeck, G. Acylated and desacyl ghrelin stimulate lipid accumulation in human visceral adipocytes. Int. J. Obes. 2009, 33, 541−552. (98) Barazzoni, R.; Zanetti, M.; Ferreira, C.; Vinci, P.; Pirulli, A.; Mucci, M.; Dore, F.; Fonda, M.; Ciocchi, B.; Cattin, L.; Guarnieri, G. Relationships between desacylated and acylated ghrelin and insulin sensitivity in the metabolic syndrome. J. Clin. Endocrinol. Metab. 2007, 92, 3935−3940. (99) Broglio, F.; Arvat, E.; Benso, A.; Gottero, C.; Muccioli, G.; Papotti, M.; van der Lely, A. J.; Deghenghi, R.; Ghigo, E. Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J. Clin. Endocrinol. Metab. 2001, 86, 5083−5086. (100) Tong, J.; Prigeon, R. L.; Davis, H. W.; Bidlingmaier, M.; Tschop, M. H.; D’Alessio, D. Physiologic concentrations of exogenously infused ghrelin reduces insulin secretion without affecting insulin sensitivity in healthy humans. J. Clin. Endocrinol. Metab. 2013, 98, 2536−2543. (101) Reimer, M. K.; Pacini, G.; Ahren, B. Dose-dependent inhibition by ghrelin of insulin secretion in the mouse. Endocrinology 2003, 144, 916−921. (102) Dezaki, K.; Sone, H.; Yada, T. Ghrelin is a physiological regulator of insulin release in pancreatic islets and glucose homeostasis. Pharmacol. Ther. 2008, 118, 239−249. (103) Vestergaard, E. T.; Gormsen, L. C.; Jessen, N.; Lund, S.; Hansen, T. K.; Moller, N.; Jorgensen, J. O. Ghrelin infusion in humans induces acute insulin resistance and lipolysis independent of growth hormone signaling. Diabetes 2008, 57, 3205−3210. (104) Nass, R.; Pezzoli, S. S.; Oliveri, M. C.; Patrie, J. T.; Harrell, F. E., Jr.; Clasey, J. L.; Heymsfield, S. B.; Bach, M. A.; Vance, M. L.; Thorner, M. O. Effects of an oral ghrelin mimetic on body composition and clinical outcomes in healthy older adults: a randomized trial. Ann. Int. Med. 2008, 149, 601−611. (105) Khojasteh-Bakht, S. C.; O’Donnell, J. P.; Fouda, H. G.; Potchoiba, M. J. Metabolism, pharmacokinetics, tissue distribution, and excretion of [14C]CP-424391 in rats. Drug Metab. Dispos. 2005, 33, 190−199. (106) Sun, Y.; Asnicar, M.; Saha, P. K.; Chan, L.; Smith, R. G. Ablation of ghrelin improves the diabetic but not obese phenotype of ob/ob mice. Cell Metab. 2006, 3, 379−386. (107) Sun, Y.; Butte, N. F.; Garcia, J. M.; Smith, R. G. Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance. Endocrinology 2008, 149, 843−850. (108) Asakawa, A.; Inui, A.; Kaga, T.; Katsuura, G.; Fujimiya, M.; Fujino, M. A.; Kasuga, M. Antagonism of ghrelin receptor reduces food intake and body weight gain in mice. Gut 2003, 52, 947−952. (109) Dezaki, K.; Hosoda, H.; Kakei, M.; Hashiguchi, S.; Watanabe, M.; Kangawa, K.; Yada, T. Endogenous ghrelin in pancreatic islets restricts insulin release by attenuating Ca2+ signaling in beta-cells: implication in the glycemic control in rodents. Diabetes 2004, 53, 3142−3151. (110) Dezaki, K.; Damdindorj, B.; Sone, H.; Dyachok, O.; Tengholm, A.; Gylfe, E.; Kurashina, T.; Yoshida, M.; Kakei, M.; Yada, T. Ghrelin attenuates cAMP-PKA signaling to evoke insulinostatic cascade in islet beta-cells. Diabetes 2011, 60, 2315−2324. (111) Liu, B.; Garcia, E. A.; Korbonits, M. Genetic studies on the ghrelin, growth hormone secretagogue receptor (GHSR) and ghrelin O-acyl transferase (GOAT) genes. Peptides 2011, 32, 2191−2207. (112) Ukkola, O. Genetic variants of ghrelin in metabolic disorders. Peptides 2011, 32, 2319−2322. (113) Gueorguiev, M.; Korbonits, M. Genetics of the ghrelin system. Endocr. Dev. 2013, 25, 25−40. (114) Speliotes, E. K.; Willer, C. J.; Berndt, S. I.; Monda, K. L.; Thorleifsson, G.; Jackson, A. U.; Lango Allen, H.; Lindgren, C. M.; Luan, J.; Magi, R.; Randall, J. C.; Vedantam, S.; Winkler, T. W.; Qi, L.;

Workalemahu, T.; Heid, I. M.; Steinthorsdottir, V.; Stringham, H. M.; Weedon, M. N.; Wheeler, E.; Wood, A. R.; Ferreira, T.; Weyant, R. J.; Segre, A. V.; Estrada, K.; Liang, L.; Nemesh, J.; Park, J. H.; Gustafsson, S.; Kilpelainen, T. O.; Yang, J.; Bouatia-Naji, N.; Esko, T.; Feitosa, M. F.; Kutalik, Z.; Mangino, M.; Raychaudhuri, S.; Scherag, A.; Smith, A. V.; Welch, R.; Zhao, J. H.; Aben, K. K.; Absher, D. M.; Amin, N.; Dixon, A. L.; Fisher, E.; Glazer, N. L.; Goddard, M. E.; Heard-Costa, N. L.; Hoesel, V.; Hottenga, J. J.; Johansson, A.; Johnson, T.; Ketkar, S.; Lamina, C.; Li, S.; Moffatt, M. F.; Myers, R. H.; Narisu, N.; Perry, J. R.; Peters, M. J.; Preuss, M.; Ripatti, S.; Rivadeneira, F.; Sandholt, C.; Scott, L. J.; Timpson, N. J.; Tyrer, J. P.; van Wingerden, S.; Watanabe, R. M.; White, C. C.; Wiklund, F.; Barlassina, C.; Chasman, D. I.; Cooper, M. N.; Jansson, J. O.; Lawrence, R. W.; Pellikka, N.; Prokopenko, I.; Shi, J.; Thiering, E.; Alavere, H.; Alibrandi, M. T.; Almgren, P.; Arnold, A. M.; Aspelund, T.; Atwood, L. D.; Balkau, B.; Balmforth, A. J.; Bennett, A. J.; Ben-Shlomo, Y.; Bergman, R. N.; Bergmann, S.; Biebermann, H.; Blakemore, A. I.; Boes, T.; Bonnycastle, L. L.; Bornstein, S. R.; Brown, M. J.; Buchanan, T. A.; Busonero, F.; Campbell, H.; Cappuccio, F. P.; Cavalcanti-Proenca, C.; Chen, Y. D.; Chen, C. M.; Chines, P. S.; Clarke, R.; Coin, L.; Connell, J.; Day, I. N.; den Heijer, M.; Duan, J.; Ebrahim, S.; Elliott, P.; Elosua, R.; Eiriksdottir, G.; Erdos, M. R.; Eriksson, J. G.; Facheris, M. F.; Felix, S. B.; Fischer-Posovszky, P.; Folsom, A. R.; Friedrich, N.; Freimer, N. B.; Fu, M.; Gaget, S.; Gejman, P. V.; Geus, E. J.; Gieger, C.; Gjesing, A. P.; Goel, A.; Goyette, P.; Grallert, H.; Grassler, J.; Greenawalt, D. M.; Groves, C. J.; Gudnason, V.; Guiducci, C.; Hartikainen, A. L.; Hassanali, N.; Hall, A. S.; Havulinna, A. S.; Hayward, C.; Heath, A. C.; Hengstenberg, C.; Hicks, A. A.; Hinney, A.; Hofman, A.; Homuth, G.; Hui, J.; Igl, W.; Iribarren, C.; Isomaa, B.; Jacobs, K. B.; Jarick, I.; Jewell, E.; John, U.; Jorgensen, T.; Jousilahti, P.; Jula, A.; Kaakinen, M.; Kajantie, E.; Kaplan, L. M.; Kathiresan, S.; Kettunen, J.; Kinnunen, L.; Knowles, J. W.; Kolcic, I.; Konig, I. R.; Koskinen, S.; Kovacs, P.; Kuusisto, J.; Kraft, P.; Kvaloy, K.; Laitinen, J.; Lantieri, O.; Lanzani, C.; Launer, L. J.; Lecoeur, C.; Lehtimaki, T.; Lettre, G.; Liu, J.; Lokki, M. L.; Lorentzon, M.; Luben, R. N.; Ludwig, B.; MAGIC; Manunta, P.; Marek, D.; Marre, M.; Martin, N. G.; McArdle, W. L.; McCarthy, A.; McKnight, B.; Meitinger, T.; Melander, O.; Meyre, D.; Midthjell, K.; Montgomery, G. W.; Morken, M. A.; Morris, A. P.; Mulic, R.; Ngwa, J. S.; Nelis, M.; Neville, M. J.; Nyholt, D. R.; O’Donnell, C. J.; O’Rahilly, S.; Ong, K. K.; Oostra, B.; Pare, G.; Parker, A. N.; Perola, M.; Pichler, I.; Pietilainen, K. H.; Platou, C. G.; Polasek, O.; Pouta, A.; Rafelt, S.; Raitakari, O.; Rayner, N. W.; Ridderstrale, M.; Rief, W.; Ruokonen, A.; Robertson, N. R.; Rzehak, P.; Salomaa, V.; Sanders, A. R.; Sandhu, M. S.; Sanna, S.; Saramies, J.; Savolainen, M. J.; Scherag, S.; Schipf, S.; Schreiber, S.; Schunkert, H.; Silander, K.; Sinisalo, J.; Siscovick, D. S.; Smit, J. H.; Soranzo, N.; Sovio, U.; Stephens, J.; Surakka, I.; Swift, A. J.; Tammesoo, M. L.; Tardif, J. C.; Teder-Laving, M.; Teslovich, T. M.; Thompson, J. R.; Thomson, B.; Tonjes, A.; Tuomi, T.; van Meurs, J. B.; van Ommen, G. J.; Vatin, V.; Viikari, J.; Visvikis-Siest, S.; Vitart, V.; Vogel, C. I.; Voight, B. F.; Waite, L. L.; Wallaschofski, H.; Walters, G. B.; Widen, E.; Wiegand, S.; Wild, S. H.; Willemsen, G.; Witte, D. R.; Witteman, J. C.; Xu, J.; Zhang, Q.; Zgaga, L.; Ziegler, A.; Zitting, P.; Beilby, J. P.; Farooqi, I. S.; Hebebrand, J.; Huikuri, H. V.; James, A. L.; Kahonen, M.; Levinson, D. F.; Macciardi, F.; Nieminen, M. S.; Ohlsson, C.; Palmer, L. J.; Ridker, P. M.; Stumvoll, M.; Beckmann, J. S.; Boeing, H.; Boerwinkle, E.; Boomsma, D. I.; Caulfield, M. J.; Chanock, S. J.; Collins, F. S.; Cupples, L. A.; Smith, G. D.; Erdmann, J.; Froguel, P.; Gronberg, H.; Gyllensten, U.; Hall, P.; Hansen, T.; Harris, T. B.; Hattersley, A. T.; Hayes, R. B.; Heinrich, J.; Hu, F. B.; Hveem, K.; Illig, T.; Jarvelin, M. R.; Kaprio, J.; Karpe, F.; Khaw, K. T.; Kiemeney, L. A.; Krude, H.; Laakso, M.; Lawlor, D. A.; Metspalu, A.; Munroe, P. B.; Ouwehand, W. H.; Pedersen, O.; Penninx, B. W.; Peters, A.; Pramstaller, P. P.; Quertermous, T.; Reinehr, T.; Rissanen, A.; Rudan, I.; Samani, N. J.; Schwarz, P. E.; Shuldiner, A. R.; Spector, T. D.; Tuomilehto, J.; Uda, M.; Uitterlinden, A.; Valle, T. T.; Wabitsch, M.; Waeber, G.; Wareham, N. J.; Watkins, H.; Procardis, C.; Wilson, J. F.; Wright, A. F.; Zillikens, M. C.; Chatterjee, N.; McCarroll, S. A.; Purcell, S.; Schadt, E. E.; Visscher, P. M.; Assimes, T. L.; Borecki, I. B.; Deloukas, P.; Fox, C. S.; Groop, L. C.; Haritunians, T.; Hunter, O

dx.doi.org/10.1021/jm5003183 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Perspective

D. J.; Kaplan, R. C.; Mohlke, K. L.; O’Connell, J. R.; Peltonen, L.; Schlessinger, D.; Strachan, D. P.; van Duijn, C. M.; Wichmann, H. E.; Frayling, T. M.; Thorsteinsdottir, U.; Abecasis, G. R.; Barroso, I.; Boehnke, M.; Stefansson, K.; North, K. E.; McCarthy, M. I.; Hirschhorn, J. N.; Ingelsson, E.; Loos, R. J. Association analyses of 249,796 individuals reveal 18 new loci associated with body mass index. Nat. Genet. 2010, 42, 937−948. (115) Berndt, S. I.; Gustafsson, S.; Magi, R.; Ganna, A.; Wheeler, E.; Feitosa, M. F.; Justice, A. E.; Monda, K. L.; Croteau-Chonka, D. C.; Day, F. R.; Esko, T.; Fall, T.; Ferreira, T.; Gentilini, D.; Jackson, A. U.; Luan, J.; Randall, J. C.; Vedantam, S.; Willer, C. J.; Winkler, T. W.; Wood, A. R.; Workalemahu, T.; Hu, Y. J.; Lee, S. H.; Liang, L.; Lin, D. Y.; Min, J. L.; Neale, B. M.; Thorleifsson, G.; Yang, J.; Albrecht, E.; Amin, N.; Bragg-Gresham, J. L.; Cadby, G.; den Heijer, M.; Eklund, N.; Fischer, K.; Goel, A.; Hottenga, J. J.; Huffman, J. E.; Jarick, I.; Johansson, A.; Johnson, T.; Kanoni, S.; Kleber, M. E.; Konig, I. R.; Kristiansson, K.; Kutalik, Z.; Lamina, C.; Lecoeur, C.; Li, G.; Mangino, M.; McArdle, W. L.; Medina-Gomez, C.; Muller-Nurasyid, M.; Ngwa, J. S.; Nolte, I. M.; Jaternoster, L.; Pechlivanis, S.; Perola, M.; Peters, M. J.; Preuss, M.; Rose, L. M.; Shi, J.; Shungin, D.; Smith, A. V.; Strawbridge, R. J.; Surakka, I.; Teumer, A.; Trip, M. D.; Tyrer, J.; Van Vliet-Ostaptchouk, J. V.; Vandenput, L.; Waite, L. L.; Zhao, J. H.; Absher, D.; Asselbergs, F. W.; Atalay, M.; Attwood, A. P.; Balmforth, A. J.; Basart, H.; Beilby, J.; Bonnycastle, L. L.; Brambilla, P.; Bruinenberg, M.; Campbell, H.; Chasman, D. I.; Chines, P. S.; Collins, F. S.; Connell, J. M.; Cookson, W. O.; de Faire, U.; de Vegt, F.; Dei, M.; Dimitriou, M.; Edkins, S.; Estrada, K.; Evans, D. M.; Farrall, M.; Ferrario, M. M.; Ferrieres, J.; Franke, L.; Frau, F.; Gejman, P. V.; Grallert, H.; Gronberg, H.; Gudnason, V.; Hall, A. S.; Hall, P.; Hartikainen, A. L.; Hayward, C.; Heard-Costa, N. L.; Heath, A. C.; Hebebrand, J.; Homuth, G.; Hu, F. B.; Hunt, S. E.; Hypponen, E.; Iribarren, C.; Jacobs, K. B.; Jansson, J. O.; Jula, A.; Kahonen, M.; Kathiresan, S.; Kee, F.; Khaw, K. T.; Kivimaki, M.; Koenig, W.; Kraja, A. T.; Kumari, M.; Kuulasmaa, K.; Kuusisto, J.; Laitinen, J. H.; Lakka, T. A.; Langenberg, C.; Launer, L. J.; Lind, L.; Lindstrom, J.; Liu, J.; Liuzzi, A.; Lokki, M. L.; Lorentzon, M.; Madden, P. A.; Magnusson, P. K.; Manunta, P.; Marek, D.; Marz, W.; Mateo Leach, I.; McKnight, B.; Medland, S. E.; Mihailov, E.; Milani, L.; Montgomery, G. W.; Mooser, V.; Muhleisen, T. W.; Munroe, P. B.; Musk, A. W.; Narisu, N.; Navis, G.; Nicholson, G.; Nohr, E. A.; Ong, K. K.; Oostra, B. A.; Palmer, C. N.; Palotie, A.; Peden, J. F.; Pedersen, N.; Peters, A.; Polasek, O.; Pouta, A.; Pramstaller, P. P.; Prokopenko, I.; Putter, C.; Radhakrishnan, A.; Raitakari, O.; Rendon, A.; Rivadeneira, F.; Rudan, I.; Saaristo, T. E.; Sambrook, J. G.; Sanders, A. R.; Sanna, S.; Saramies, J.; Schipf, S.; Schreiber, S.; Schunkert, H.; Shin, S. Y.; Signorini, S.; Sinisalo, J.; Skrobek, B.; Soranzo, N.; Stancakova, A.; Stark, K.; Stephens, J. C.; Stirrups, K.; Stolk, R. P.; Stumvoll, M.; Swift, A. J.; Theodoraki, E. V.; Thorand, B.; Tregouet, D. A.; Tremoli, E.; Van der Klauw, M. M.; van Meurs, J. B.; Vermeulen, S. H.; Viikari, J.; Virtamo, J.; Vitart, V.; Waeber, G.; Wang, Z.; Widen, E.; Wild, S. H.; Willemsen, G.; Winkelmann, B. R.; Witteman, J. C.; Wolffenbuttel, B. H.; Wong, A.; Wright, A. F.; Zillikens, M. C.; Amouyel, P.; Boehm, B. O.; Boerwinkle, E.; Boomsma, D. I.; Caulfield, M. J.; Chanock, S. J.; Cupples, L. A.; Cusi, D.; Dedoussis, G. V.; Erdmann, J.; Eriksson, J. G.; Franks, P. W.; Froguel, P.; Gieger, C.; Gyllensten, U.; Hamsten, A.; Harris, T. B.; Hengstenberg, C.; Hicks, A. A.; Hingorani, A.; Hinney, A.; Hofman, A.; Hovingh, K. G.; Hveem, K.; Illig, T.; Jarvelin, M. R.; Jockel, K. H.; Keinanen-Kiukaanniemi, S. M.; Kiemeney, L. A.; Kuh, D.; Laakso, M.; Lehtimaki, T.; Levinson, D. F.; Martin, N. G.; Metspalu, A.; Morris, A. D.; Nieminen, M. S.; Njolstad, I.; Ohlsson, C.; Oldehinkel, A. J.; Ouwehand, W. H.; Palmer, L. J.; Penninx, B.; Power, C.; Province, M. A.; Psaty, B. M.; Qi, L.; Rauramaa, R.; Ridker, P. M.; Ripatti, S.; Salomaa, V.; Samani, N. J.; Snieder, H.; Sorensen, T. I.; Spector, T. D.; Stefansson, K.; Tonjes, A.; Tuomilehto, J.; Uitterlinden, A. G.; Uusitupa, M.; van der Harst, P.; Vollenweider, P.; Wallaschofski, H.; Wareham, N. J.; Watkins, H.; Wichmann, H. E.; Wilson, J. F.; Abecasis, G. R.; Assimes, T. L.; Barroso, I.; Boehnke, M.; Borecki, I. B.; Deloukas, P.; Fox, C. S.; Frayling, T.; Groop, L. C.; Haritunian, T.; Heid, I. M.; Hunter, D.; Kaplan, R. C.; Karpe, F.; Moffatt, M. F.;

Mohlke, K. L.; O’Connell, J. R.; Pawitan, Y.; Schadt, E. E.; Schlessinger, D.; Steinthorsdottir, V.; Strachan, D. P.; Thorsteinsdottir, U.; van Duijn, C. M.; Visscher, P. M.; Di Blasio, A. M.; Hirschhorn, J. N.; Lindgren, C. M.; Morris, A. P.; Meyre, D.; Scherag, A.; McCarthy, M. I.; Speliotes, E. K.; North, K. E.; Loos, R. J.; Ingelsson, E. Genome-wide meta-analysis identifies 11 new loci for anthropometric traits and provides insights into genetic architecture. Nat. Genet. 2013, 45, 501−512. (116) Saxena, R.; Hivert, M. F.; Langenberg, C.; Tanaka, T.; Pankow, J. S.; Vollenweider, P.; Lyssenko, V.; Bouatia-Naji, N.; Dupuis, J.; Jackson, A. U.; Kao, W. H.; Li, M.; Glazer, N. L.; Manning, A. K.; Luan, J.; Stringham, H. M.; Prokopenko, I.; Johnson, T.; Grarup, N.; Boesgaard, T. W.; Lecoeur, C.; Shrader, P.; O’Connell, J.; Ingelsson, E.; Couper, D. J.; Rice, K.; Song, K.; Andreasen, C. H.; Dina, C.; Kottgen, A.; Le Bacquer, O.; Pattou, F.; Taneera, J.; Steinthorsdottir, V.; Rybin, D.; Ardlie, K.; Sampson, M.; Qi, L.; van Hoek, M.; Weedon, M. N.; Aulchenko, Y. S.; Voight, B. F.; Grallert, H.; Balkau, B.; Bergman, R. N.; Bielinski, S. J.; Bonnefond, A.; Bonnycastle, L. L.; Borch-Johnsen, K.; Bottcher, Y.; Brunner, E.; Buchanan, T. A.; Bumpstead, S. J.; Cavalcanti-Proenca, C.; Charpentier, G.; Chen, Y. D.; Chines, P. S.; Collins, F. S.; Cornelis, M.; G, J. C.; Delplanque, J.; Doney, A.; Egan, J. M.; Erdos, M. R.; Firmann, M.; Forouhi, N. G.; Fox, C. S.; Goodarzi, M. O.; Graessler, J.; Hingorani, A.; Isomaa, B.; Jorgensen, T.; Kivimaki, M.; Kovacs, P.; Krohn, K.; Kumari, M.; Lauritzen, T.; Levy-Marchal, C.; Mayor, V.; McAteer, J. B.; Meyre, D.; Mitchell, B. D.; Mohlke, K. L.; Morken, M. A.; Narisu, N.; Palmer, C. N.; Pakyz, R.; Pascoe, L.; Payne, F.; Pearson, D.; Rathmann, W.; Sandbaek, A.; Sayer, A. A.; Scott, L. J.; Sharp, S. J.; Sijbrands, E.; Singleton, A.; Siscovick, D. S.; Smith, N. L.; Sparso, T.; Swift, A. J.; Syddall, H.; Thorleifsson, G.; Tonjes, A.; Tuomi, T.; Tuomilehto, J.; Valle, T. T.; Waeber, G.; Walley, A.; Waterworth, D. M.; Zeggini, E.; Zhao, J. H.; GIANT Consortium; Illig, T.; Wichmann, H. E.; Wilson, J. F.; van Duijn, C.; Hu, F. B.; Morris, A. D.; Frayling, T. M.; Hattersley, A. T.; Thorsteinsdottir, U.; Stefansson, K.; Nilsson, P.; Syvanen, A. C.; Shuldiner, A. R.; Walker, M.; Bornstein, S. R.; Schwarz, P.; Williams, G. H.; Nathan, D. M.; Kuusisto, J.; Laakso, M.; Cooper, C.; Marmot, M.; Ferrucci, L.; Mooser, V.; Stumvoll, M.; Loos, R. J.; Altshuler, D.; Psaty, B. M.; Rotter, J. I.; Boerwinkle, E.; Hansen, T.; Pedersen, O.; Florez, J. C.; McCarthy, M. I.; Boehnke, M.; Barroso, I.; Sladek, R.; Froguel, P.; Meigs, J. B.; Groop, L.; Wareham, N. J.; Watanabe, R. M. Genetic variation in GIPR influences the glucose and insulin responses to an oral glucose challenge. Nat. Genet. 2010, 42, 142−148. (117) Dupuis, J.; Langenberg, C.; Prokopenko, I.; Saxena, R.; Soranzo, N.; Jackson, A. U.; Wheeler, E.; Glazer, N. L.; Bouatia-Naji, N.; Gloyn, A. L.; Lindgren, C. M.; Magi, R.; Morris, A. P.; Randall, J.; Johnson, T.; Elliott, P.; Rybin, D.; Thorleifsson, G.; Steinthorsdottir, V.; Henneman, P.; Grallert, H.; Dehghan, A.; Hottenga, J. J.; Franklin, C. S.; Navarro, P.; Song, K.; Goel, A.; Perry, J. R.; Egan, J. M.; Lajunen, T.; Grarup, N.; Sparso, T.; Doney, A.; Voight, B. F.; Stringham, H. M.; Li, M.; Kanoni, S.; Shrader, P.; Cavalcanti-Proenca, C.; Kumari, M.; Qi, L.; Timpson, N. J.; Gieger, C.; Zabena, C.; Rocheleau, G.; Ingelsson, E.; An, P.; O’Connell, J.; Luan, J.; Elliott, A.; McCarroll, S. A.; Payne, F.; Roccasecca, R. M.; Pattou, F.; Sethupathy, P.; Ardlie, K.; Ariyurek, Y.; Balkau, B.; Barter, P.; Beilby, J. P.; Ben-Shlomo, Y.; Benediktsson, R.; Bennett, A. J.; Bergmann, S.; Bochud, M.; Boerwinkle, E.; Bonnefond, A.; Bonnycastle, L. L.; Borch-Johnsen, K.; Bottcher, Y.; Brunner, E.; Bumpstead, S. J.; Charpentier, G.; Chen, Y. D.; Chines, P.; Clarke, R.; Coin, L. J.; Cooper, M. N.; Cornelis, M.; Crawford, G.; Crisponi, L.; Day, I. N.; de Geus, E. J.; Delplanque, J.; Dina, C.; Erdos, M. R.; Fedson, A. C.; Fischer-Rosinsky, A.; Forouhi, N. G.; Fox, C. S.; Frants, R.; Franzosi, M. G.; Galan, P.; Goodarzi, M. O.; Graessler, J.; Groves, C. J.; Grundy, S.; Gwilliam, R.; Gyllensten, U.; Hadjadj, S.; Hallmans, G.; Hammond, N.; Han, X.; Hartikainen, A. L.; Hassanali, N.; Hayward, C.; Heath, S. C.; Hercberg, S.; Herder, C.; Hicks, A. A.; Hillman, D. R.; Hingorani, A. D.; Hofman, A.; Hui, J.; Hung, J.; Isomaa, B.; Johnson, P. R.; Jorgensen, T.; Jula, A.; Kaakinen, M.; Kaprio, J.; Kesaniemi, Y. A.; Kivimaki, M.; Knight, B.; Koskinen, S.; Kovacs, P.; Kyvik, K. O.; Lathrop, G. M.; Lawlor, D. A.; Le Bacquer, O.; Lecoeur, C.; Li, Y.; Lyssenko, V.; Mahley, R.; Mangino, M.; P

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Manning, A. K.; Martinez-Larrad, M. T.; McAteer, J. B.; McCulloch, L. J.; McPherson, R.; Meisinger, C.; Melzer, D.; Meyre, D.; Mitchell, B. D.; Morken, M. A.; Mukherjee, S.; Naitza, S.; Narisu, N.; Neville, M. J.; Oostra, B. A.; Orru, M.; Pakyz, R.; Palmer, C. N.; Paolisso, G.; Pattaro, C.; Pearson, D.; Peden, J. F.; Pedersen, N. L.; Perola, M.; Pfeiffer, A. F.; Pichler, I.; Polasek, O.; Posthuma, D.; Potter, S. C.; Pouta, A.; Province, M. A.; Psaty, B. M.; Rathmann, W.; Rayner, N. W.; Rice, K.; Ripatti, S.; Rivadeneira, F.; Roden, M.; Rolandsson, O.; Sandbaek, A.; Sandhu, M.; Sanna, S.; Sayer, A. A.; Scheet, P.; Scott, L. J.; Seedorf, U.; Sharp, S. J.; Shields, B.; Sigurethsson, G.; Sijbrands, E. J.; Silveira, A.; Simpson, L.; Singleton, A.; Smith, N. L.; Sovio, U.; Swift, A.; Syddall, H.; Syvanen, A. C.; Tanaka, T.; Thorand, B.; Tichet, J.; Tonjes, A.; Tuomi, T.; Uitterlinden, A. G.; van Dijk, K. W.; van Hoek, M.; Varma, D.; Visvikis-Siest, S.; Vitart, V.; Vogelzangs, N.; Waeber, G.; Wagner, P. J.; Walley, A.; Walters, G. B.; Ward, K. L.; Watkins, H.; Weedon, M. N.; Wild, S. H.; Willemsen, G.; Witteman, J. C.; Yarnell, J. W.; Zeggini, E.; Zelenika, D.; Zethelius, B.; Zhai, G.; Zhao, J. H.; Zillikens, M. C.; DIAGRAM Consortium; GIANT Consortium; Global BPgen Consortium; Borecki, I. B.; Loos, R. J.; Meneton, P.; Magnusson, P. K.; Nathan, D. M.; Williams, G. H.; Hattersley, A. T.; Silander, K.; Salomaa, V.; Smith, G. D.; Bornstein, S. R.; Schwarz, P.; Spranger, J.; Karpe, F.; Shuldiner, A. R.; Cooper, C.; Dedoussis, G. V.; SerranoRios, M.; Morris, A. D.; Lind, L.; Palmer, L. J.; Hu, F. B.; Franks, P. W.; Ebrahim, S.; Marmot, M.; Kao, W. H.; Pankow, J. S.; Sampson, M. J.; Kuusisto, J.; Laakso, M.; Hansen, T.; Pedersen, O.; Pramstaller, P. P.; Wichmann, H. E.; Illig, T.; Rudan, I.; Wright, A. F.; Stumvoll, M.; Campbell, H.; Wilson, J. F.; Anders Hamsten on behalf of Procardis Consortium; Bergman, R. N.; Buchanan, T. A.; Collins, F. S.; Mohlke, K. L.; Tuomilehto, J.; Valle, T. T.; Altshuler, D.; Rotter, J. I.; Siscovick, D. S.; Penninx, B. W.; Boomsma, D. I.; Deloukas, P.; Spector, T. D.; Frayling, T. M.; Ferrucci, L.; Kong, A.; Thorsteinsdottir, U.; Stefansson, K.; van Duijn, C. M.; Aulchenko, Y. S.; Cao, A.; Scuteri, A.; Schlessinger, D.; Uda, M.; Ruokonen, A.; Jarvelin, M. R.; Waterworth, D. M.; Vollenweider, P.; Peltonen, L.; Mooser, V.; Abecasis, G. R.; Wareham, N. J.; Sladek, R.; Froguel, P.; Watanabe, R. M.; Meigs, J. B.; Groop, L.; Boehnke, M.; McCarthy, M. I.; Florez, J. C.; Barroso, I. New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk. Nat. Genet. 2010, 42, 105−116. (118) Voight, B. F.; Scott, L. J.; Steinthorsdottir, V.; Morris, A. P.; Dina, C.; Welch, R. P.; Zeggini, E.; Huth, C.; Aulchenko, Y. S.; Thorleifsson, G.; McCulloch, L. J.; Ferreira, T.; Grallert, H.; Amin, N.; Wu, G.; Willer, C. J.; Raychaudhuri, S.; McCarroll, S. A.; Langenberg, C.; Hofmann, O. M.; Dupuis, J.; Qi, L.; Segre, A. V.; van Hoek, M.; Navarro, P.; Ardlie, K.; Balkau, B.; Benediktsson, R.; Bennett, A. J.; Blagieva, R.; Boerwinkle, E.; Bonnycastle, L. L.; Bengtsson Bostrom, K.; Bravenboer, B.; Bumpstead, S.; Burtt, N. P.; Charpentier, G.; Chines, P. S.; Cornelis, M.; Couper, D. J.; Crawford, G.; Doney, A. S.; Elliott, K. S.; Elliott, A. L.; Erdos, M. R.; Fox, C. S.; Franklin, C. S.; Ganser, M.; Gieger, C.; Grarup, N.; Green, T.; Griffin, S.; Groves, C. J.; Guiducci, C.; Hadjadj, S.; Hassanali, N.; Herder, C.; Isomaa, B.; Jackson, A. U.; Johnson, P. R.; Jorgensen, T.; Kao, W. H.; Klopp, N.; Kong, A.; Kraft, P.; Kuusisto, J.; Lauritzen, T.; Li, M.; Lieverse, A.; Lindgren, C. M.; Lyssenko, V.; Marre, M.; Meitinger, T.; Midthjell, K.; Morken, M. A.; Narisu, N.; Nilsson, P.; Owen, K. R.; Payne, F.; Perry, J. R.; Petersen, A. K.; Platou, C.; Proenca, C.; Prokopenko, I.; Rathmann, W.; Rayner, N. W.; Robertson, N. R.; Rocheleau, G.; Roden, M.; Sampson, M. J.; Saxena, R.; Shields, B. M.; Shrader, P.; Sigurdsson, G.; Sparso, T.; Strassburger, K.; Stringham, H. M.; Sun, Q.; Swift, A. J.; Thorand, B.; Tichet, J.; Tuomi, T.; van Dam, R. M.; van Haeften, T. W.; van Herpt, T.; van Vliet-Ostaptchouk, J. V.; Walters, G. B.; Weedon, M. N.; Wijmenga, C.; Witteman, J.; The MAGIC investigators; The GIANT Consortium; Bergman, R. N.; Cauchi, S.; Collins, F. S.; Gloyn, A. L.; Gyllensten, U.; Hansen, T.; Hide, W. A.; Hitman, G. A.; Hofman, A.; Hunter, D. J.; Hveem, K.; Laakso, M.; Mohlke, K. L.; Morris, A. D.; Palmer, C. N.; Pramstaller, P. P.; Rudan, I.; Sijbrands, E.; Stein, L. D.; Tuomilehto, J.; Uitterlinden, A.; Walker, M.; Wareham, N. J.; Watanabe, R. M.; Abecasis, G. R.; Boehm, B. O.; Campbell, H.; Daly, M. J.; Hattersley, A. T.; Hu, F. B.;

Meigs, J. B.; Pankow, J. S.; Pedersen, O.; Wichmann, H. E.; Barroso, I.; Florez, J. C.; Frayling, T. M.; Groop, L.; Sladek, R.; Thorsteinsdottir, U.; Wilson, J. F.; Illig, T.; Froguel, P.; van Duijn, C. M.; Stefansson, K.; Altshuler, D.; Boehnke, M.; McCarthy, M. I. Twelve type 2 diabetes susceptibility loci identified through large-scale association analysis. Nat. Genet. 2010, 42, 579−589. (119) Soranzo, N.; Sanna, S.; Wheeler, E.; Gieger, C.; Radke, D.; Dupuis, J.; Bouatia-Naji, N.; Langenberg, C.; Prokopenko, I.; Stolerman, E.; Sandhu, M. S.; Heeney, M. M.; Devaney, J. M.; Reilly, M. P.; Ricketts, S. L.; Stewart, A. F.; Voight, B. F.; Willenborg, C.; Wright, B.; Altshuler, D.; Arking, D.; Balkau, B.; Barnes, D.; Boerwinkle, E.; Bohm, B.; Bonnefond, A.; Bonnycastle, L. L.; Boomsma, D. I.; Bornstein, S. R.; Bottcher, Y.; Bumpstead, S.; Burnett-Miller, M. S.; Campbell, H.; Cao, A.; Chambers, J.; Clark, R.; Collins, F. S.; Coresh, J.; de Geus, E. J.; Dei, M.; Deloukas, P.; Doring, A.; Egan, J. M.; Elosua, R.; Ferrucci, L.; Forouhi, N.; Fox, C. S.; Franklin, C.; Franzosi, M. G.; Gallina, S.; Goel, A.; Graessler, J.; Grallert, H.; Greinacher, A.; Hadley, D.; Hall, A.; Hamsten, A.; Hayward, C.; Heath, S.; Herder, C.; Homuth, G.; Hottenga, J. J.; Hunter-Merrill, R.; Illig, T.; Jackson, A. U.; Jula, A.; Kleber, M.; Knouff, C. W.; Kong, A.; Kooner, J.; Kottgen, A.; Kovacs, P.; Krohn, K.; Kuhnel, B.; Kuusisto, J.; Laakso, M.; Lathrop, M.; Lecoeur, C.; Li, M.; Li, M.; Loos, R. J.; Luan, J.; Lyssenko, V.; Magi, R.; Magnusson, P. K.; Malarstig, A.; Mangino, M.; Martinez-Larrad, M. T.; Marz, W.; McArdle, W. L.; McPherson, R.; Meisinger, C.; Meitinger, T.; Melander, O.; Mohlke, K. L.; Mooser, V. E.; Morken, M. A.; Narisu, N.; Nathan, D. M.; Nauck, M.; O’Donnell, C.; Oexle, K.; Olla, N.; Pankow, J. S.; Payne, F.; Peden, J. F.; Pedersen, N. L.; Peltonen, L.; Perola, M.; Polasek, O.; Porcu, E.; Rader, D. J.; Rathmann, W.; Ripatti, S.; Rocheleau, G.; Roden, M.; Rudan, I.; Salomaa, V.; Saxena, R.; Schlessinger, D.; Schunkert, H.; Schwarz, P.; Seedorf, U.; Selvin, E.; Serrano-Rios, M.; Shrader, P.; Silveira, A.; Siscovick, D.; Song, K.; Spector, T. D.; Stefansson, K.; Steinthorsdottir, V.; Strachan, D. P.; Strawbridge, R.; Stumvoll, M.; Surakka, I.; Swift, A. J.; Tanaka, T.; Teumer, A.; Thorleifsson, G.; Thorsteinsdottir, U.; Tonjes, A.; Usala, G.; Vitart, V.; Volzke, H.; Wallaschofski, H.; Waterworth, D. M.; Watkins, H.; Wichmann, H. E.; Wild, S. H.; Willemsen, G.; Williams, G. H.; Wilson, J. F.; Winkelmann, J.; Wright, A. F.; WTCCC; Zabena, C.; Zhao, J. H.; Epstein, S. E.; Erdmann, J.; Hakonarson, H. H.; Kathiresan, S.; Khaw, K. T.; Roberts, R.; Samani, N. J.; Fleming, M. D.; Sladek, R.; Abecasis, G.; Boehnke, M.; Froguel, P.; Groop, L.; McCarthy, M. I.; Kao, W. H.; Florez, J. C.; Uda, M.; Wareham, N. J.; Barroso, I.; Meigs, J. B. Common variants at 10 genomic loci influence hemoglobin A(1)(C) levels via glycemic and nonglycemic pathways. Diabetes 2010, 59, 3229−3239. (120) International Consortium for Blood Pressure Genome-Wide Association Studies; Ehret, G. B.; Munroe, P. B.; Rice, K. M.; Bochud, M.; Johnson, A. D.; Chasman, D. I.; Smith, A. V.; Tobin, M. D.; Verwoert, G. C.; Hwang, S. J.; Pihur, V.; Vollenweider, P.; O’Reilly, P. F.; Amin, N.; Bragg-Gresham, J. L.; Teumer, A.; Glazer, N. L.; Launer, L.; Zhao, J. H.; Aulchenko, Y.; Heath, S.; Sober, S.; Parsa, A.; Luan, J.; Arora, P.; Dehghan, A.; Zhang, F.; Lucas, G.; Hicks, A. A.; Jackson, A. U.; Peden, J. F.; Tanaka, T.; Wild, S. H.; Rudan, I.; Igl, W.; Milaneschi, Y.; Parker, A. N.; Fava, C.; Chambers, J. C.; Fox, E. R.; Kumari, M.; Go, M. J.; van der Harst, P.; Kao, W. H.; Sjogren, M.; Vinay, D. G.; Alexander, M.; Tabara, Y.; Shaw-Hawkins, S.; Whincup, P. H.; Liu, Y.; Shi, G.; Kuusisto, J.; Tayo, B.; Seielstad, M.; Sim, X.; Nguyen, K. D.; Lehtimaki, T.; Matullo, G.; Wu, Y.; Gaunt, T. R.; Onland-Moret, N. C.; Cooper, M. N.; Platou, C. G.; Org, E.; Hardy, R.; Dahgam, S.; Palmen, J.; Vitart, V.; Braund, P. S.; Kuznetsova, T.; Uiterwaal, C. S.; Adeyemo, A.; Palmas, W.; Campbell, H.; Ludwig, B.; Tomaszewski, M.; Tzoulaki, I.; Palmer, N. D.; CARDIoGRAM Consortium; CDKGen Consortium; KidneyGen Consortium; EchoGen Consortium; CHARGE-HF Consortium; Aspelund, T.; Garcia, M.; Chang, Y. P.; O’Connell, J. R.; Steinle, N. I.; Grobbee, D. E.; Arking, D. E.; Kardia, S. L.; Morrison, A. C.; Hernandez, D.; Najjar, S.; McArdle, W. L.; Hadley, D.; Brown, M. J.; Connell, J. M.; Hingorani, A. D.; Day, I. N.; Lawlor, D. A.; Beilby, J. P.; Lawrence, R. W.; Clarke, R.; Hopewell, J. C.; Ongen, H.; Dreisbach, A. W.; Li, Y.; Young, J. H.; Q

dx.doi.org/10.1021/jm5003183 | J. Med. Chem. XXXX, XXX, XXX−XXX

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Prospective Investigation into Cancer and Nutrition (EPIC). Carcinogenesis 2008, 29, 1360−1366. (123) Martin, G. R.; Loredo, J. C.; Sun, G. Lack of association of ghrelin precursor gene variants and percentage body fat or serum lipid profiles. Obesity 2008, 16, 908−912. (124) Liao, N.; Xie, Z. K.; Huang, J.; Xie, Z. F. Association between the ghrelin Leu72Met polymorphism and type 2 diabetes risk: a metaanalysis. Gene 2013, 517, 179−183. (125) Steinle, N. I.; Pollin, T. I.; O’Connell, J. R.; Mitchell, B. D.; Shuldiner, A. R. Variants in the ghrelin gene are associated with metabolic syndrome in the Old Order Amish. J. Clin. Endocrinol. Metab. 2005, 90, 6672−6677. (126) Larsen, L. H.; Angquist, L.; Vimaleswaran, K. S.; Hager, J.; Viguerie, N.; Loos, R. J.; Handjieva-Darlenska, T.; Jebb, S. A.; Kunesova, M.; Larsen, T. M.; Martinez, J. A.; Papadaki, A.; Pfeiffer, A. F.; van Baak, M. A.; Sorensen, T.; Holst, C.; Langin, D.; Astrup, A.; Saris, W. H. Analyses of single nucleotide polymorphisms in selected nutrient-sensitive genes in weight-regain prevention: the DIOGENES study. Am. J. Clin. Nutr. 2012, 95, 1254−1260. (127) Bing, C.; Ambye, L.; Fenger, M.; Jorgensen, T.; Borch-Johnsen, K.; Madsbad, S.; Urhammer, S. A. Large-scale studies of the Leu72Met polymorphism of the ghrelin gene in relation to the metabolic syndrome and associated quantitative traits. Diabetic Med. 2005, 22, 1157−1160. (128) Garcia, E. A.; King, P.; Sidhu, K.; Ohgusu, H.; Walley, A.; Lecoeur, C.; Gueorguiev, M.; Khalaf, S.; Davies, D.; Grossman, A. B.; Kojima, M.; Petersenn, S.; Froguel, P.; Korbonits, M. The role of ghrelin and ghrelin-receptor gene variants and promoter activity in type 2 diabetes. Eur. J. Endocrinol. 2009, 161, 307−315. (129) Poykko, S.; Ukkola, O.; Kauma, H.; Savolainen, M. J.; Kesaniemi, Y. A. Ghrelin Arg51Gln mutation is a risk factor for Type 2 diabetes and hypertension in a random sample of middle-aged subjects. Diabetologia 2003, 46, 455−458. (130) Berthold, H. K.; Giannakidou, E.; Krone, W.; Mantzoros, C. S.; Gouni-Berthold, I. The Leu72Met polymorphism of the ghrelin gene is associated with a decreased risk for type 2 diabetes. Clin. Chim. Acta 2009, 399, 112−116. (131) Mager, U.; Kolehmainen, M.; Lindstrom, J.; Eriksson, J. G.; Valle, T. T.; Hamalainen, H.; Ilanne-Parikka, P.; KeinanenKiukaanniemi, S.; Tuomilehto, J. O.; Pulkkinen, L.; Uusitupa, M. I.; Finnish Diabetes Prevention Study Group.. Association between ghrelin gene variations and blood pressure in subjects with impaired glucose tolerance. Am. J. Hypertens. 2006, 19, 920−926. (132) Matzko, M. E.; Argyropoulos, G.; Wood, G. C.; Chu, X.; McCarter, R. J.; Still, C. D.; Gerhard, G. S. Association of ghrelin receptor promoter polymorphisms with weight loss following Rouxen-Y gastric bypass surgery. Obes. Surg. 2012, 22, 783−790. (133) Gjesing, A. P.; Larsen, L. H.; Torekov, S. S.; Hainerova, I. A.; Kapur, R.; Johansen, A.; Albrechtsen, A.; Boj, S.; Holst, B.; Harper, A.; Urhammer, S. A.; Borch-Johnsen, K.; Pisinger, C.; Echwald, S. M.; Eiberg, H.; Astrup, A.; Lebl, J.; Ferrer, J.; Schwartz, T. W.; Hansen, T.; Pedersen, O. Family and population-based studies of variation within the ghrelin receptor locus in relation to measures of obesity. PLoS One 2010, 5, e10084. (134) Mager, U.; Degenhardt, T.; Pulkkinen, L.; Kolehmainen, M.; Tolppanen, A. M.; Lindstrom, J.; Eriksson, J. G.; Carlberg, C.; Tuomilehto, J.; Uusitupa, M.; Finnish Diabetes Prevention Study Group.. Variations in the ghrelin receptor gene associate with obesity and glucose metabolism in individuals with impaired glucose tolerance. PLoS One 2008, 3, e2941. (135) Miyasaka, K.; Hosoya, H.; Sekime, A.; Ohta, M.; Amono, H.; Matsushita, S.; Suzuki, K.; Higuchi, S.; Funakoshi, A. Association of ghrelin receptor gene polymorphism with bulimia nervosa in a Japanese population. J. Neural Transm. 2006, 113, 1279−1285. (136) Baessler, A.; Kwitek, A. E.; Fischer, M.; Koehler, M.; Reinhard, W.; Erdmann, J.; Riegger, G.; Doering, A.; Schunkert, H.; Hengstenberg, C. Association of the ghrelin receptor gene region with left ventricular hypertrophy in the general population: results of

Bis, J. C.; Kahonen, M.; Viikari, J.; Adair, L. S.; Lee, N. R.; Chen, M. H.; Olden, M.; Pattaro, C.; Bolton, J. A.; Kottgen, A.; Bergmann, S.; Mooser, V.; Chaturvedi, N.; Frayling, T. M.; Islam, M.; Jafar, T. H.; Erdmann, J.; Kulkarni, S. R.; Bornstein, S. R.; Grassler, J.; Groop, L.; Voight, B. F.; Kettunen, J.; Howard, P.; Taylor, A.; Guarrera, S.; Ricceri, F.; Emilsson, V.; Plump, A.; Barroso, I.; Khaw, K. T.; Weder, A. B.; Hunt, S. C.; Sun, Y. V.; Bergman, R. N.; Collins, F. S.; Bonnycastle, L. L.; Scott, L. J.; Stringham, H. M.; Peltonen, L.; Perola, M.; Vartiainen, E.; Brand, S. M.; Staessen, J. A.; Wang, T. J.; Burton, P. R.; Soler Artigas, M.; Dong, Y.; Snieder, H.; Wang, X.; Zhu, H.; Lohman, K. K.; Rudock, M. E.; Heckbert, S. R.; Smith, N. L.; Wiggins, K. L.; Doumatey, A.; Shriner, D.; Veldre, G.; Viigimaa, M.; Kinra, S.; Prabhakaran, D.; Tripathy, V.; Langefeld, C. D.; Rosengren, A.; Thelle, D. S.; Corsi, A. M.; Singleton, A.; Forrester, T.; Hilton, G.; McKenzie, C. A.; Salako, T.; Iwai, N.; Kita, Y.; Ogihara, T.; Ohkubo, T.; Okamura, T.; Ueshima, H.; Umemura, S.; Eyheramendy, S.; Meitinger, T.; Wichmann, H. E.; Cho, Y. S.; Kim, H. L.; Lee, J. Y.; Scott, J.; Sehmi, J. S.; Zhang, W.; Hedblad, B.; Nilsson, P.; Smith, G. D.; Wong, A.; Narisu, N.; Stancakova, A.; Raffel, L. J.; Yao, J.; Kathiresan, S.; O’Donnell, C. J.; Schwartz, S. M.; Ikram, M. A.; Longstreth, W. T., Jr.; Mosley, T. H.; Seshadri, S.; Shrine, N. R.; Wain, L. V.; Morken, M. A.; Swift, A. J.; Laitinen, J.; Prokopenko, I.; Zitting, P.; Cooper, J. A.; Humphries, S. E.; Danesh, J.; Rasheed, A.; Goel, A.; Hamsten, A.; Watkins, H.; Bakker, S. J.; van Gilst, W. H.; Janipalli, C. S.; Mani, K. R.; Yajnik, C. S.; Hofman, A.; Mattace-Raso, F. U.; Oostra, B. A.; Demirkan, A.; Isaacs, A.; Rivadeneira, F.; Lakatta, E. G.; Orru, M.; Scuteri, A.; Ala-Korpela, M.; Kangas, A. J.; Lyytikainen, L. P.; Soininen, P.; Tukiainen, T.; Wurtz, P.; Ong, R. T.; Dorr, M.; Kroemer, H. K.; Volker, U.; Volzke, H.; Galan, P.; Hercberg, S.; Lathrop, M.; Zelenika, D.; Deloukas, P.; Mangino, M.; Spector, T. D.; Zhai, G.; Meschia, J. F.; Nalls, M. A.; Sharma, P.; Terzic, J.; Kumar, M. V.; Denniff, M.; Zukowska-Szczechowska, E.; Wagenknecht, L. E.; Fowkes, F. G.; Charchar, F. J.; Schwarz, P. E.; Hayward, C.; Guo, X.; Rotimi, C.; Bots, M. L.; Brand, E.; Samani, N. J.; Polasek, O.; Talmud, P. J.; Nyberg, F.; Kuh, D.; Laan, M.; Hveem, K.; Palmer, L. J.; van der Schouw, Y. T.; Casas, J. P.; Mohlke, K. L.; Vineis, P.; Raitakari, O.; Ganesh, S. K.; Wong, T. Y.; Tai, E. S.; Cooper, R. S.; Laakso, M.; Rao, D. C.; Harris, T. B.; Morris, R. W.; Dominiczak, A. F.; Kivimaki, M.; Marmot, M. G.; Miki, T.; Saleheen, D.; Chandak, G. R.; Coresh, J.; Navis, G.; Salomaa, V.; Han, B. G.; Zhu, X.; Kooner, J. S.; Melander, O.; Ridker, P. M.; Bandinelli, S.; Gyllensten, U. B.; Wright, A. F.; Wilson, J. F.; Ferrucci, L.; Farrall, M.; Tuomilehto, J.; Pramstaller, P. P.; Elosua, R.; Soranzo, N.; Sijbrands, E. J.; Altshuler, D.; Loos, R. J.; Shuldiner, A. R.; Gieger, C.; Meneton, P.; Uitterlinden, A. G.; Wareham, N. J.; Gudnason, V.; Rotter, J. I.; Rettig, R.; Uda, M.; Strachan, D. P.; Witteman, J. C.; Hartikainen, A. L.; Beckmann, J. S.; Boerwinkle, E.; Vasan, R. S.; Boehnke, M.; Larson, M. G.; Jarvelin, M. R.; Psaty, B. M.; Abecasis, G. R.; Chakravarti, A.; Elliott, P.; van Duijn, C. M.; Newton-Cheh, C.; Levy, D.; Caulfield, M. J.; Johnson, T. Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk. Nature 2011, 478, 103−109. (121) Middelberg, R. P.; Ferreira, M. A.; Henders, A. K.; Heath, A. C.; Madden, P. A.; Montgomery, G. W.; Martin, N. G.; Whitfield, J. B. Genetic variants in LPL, OASL and TOMM40/APOE-C1-C2-C4 genes are associated with multiple cardiovascular-related traits. BMC Med. Genet. 2011, 12, 123. (122) Dossus, L.; McKay, J. D.; Canzian, F.; Wilkening, S.; Rinaldi, S.; Biessy, C.; Olsen, A.; Tjonneland, A.; Jakobsen, M. U.; Overvad, K.; Clavel-Chapelon, F.; Boutron-Ruault, M. C.; Fournier, A.; Linseisen, J.; Lukanova, A.; Boeing, H.; Fisher, E.; Trichopoulou, A.; Georgila, C.; Trichopoulos, D.; Palli, D.; Krogh, V.; Tumino, R.; Vineis, P.; Quiros, J. R.; Sala, N.; Martinez-Garcia, C.; Dorronsoro, M.; Chirlaque, M. D.; Barricarte, A.; van Duijnhoven, F. J.; Bueno-de-Mesquita, H. B.; van Gils, C. H.; Peeters, P. H.; Hallmans, G.; Lenner, P.; Bingham, S.; Khaw, K. T.; Key, T. J.; Travis, R. C.; Ferrari, P.; Jenab, M.; Riboli, E.; Kaaks, R. Polymorphisms of genes coding for ghrelin and its receptor in relation to anthropometry, circulating levels of IGF-I and IGFBP-3, and breast cancer risk: a case-control study nested within the European R

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Journal of Medicinal Chemistry

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the MONICA/KORA Augsburg echocardiographic substudy. Hypertension 2006, 47, 920−927. (137) Baessler, A.; Fischer, M.; Mayer, B.; Koehler, M.; Wiedmann, S.; Stark, K.; Doering, A.; Erdmann, J.; Riegger, G.; Schunkert, H.; Kwitek, A. E.; Hengstenberg, C. Epistatic interaction between haplotypes of the ghrelin ligand and receptor genes influence susceptibility to myocardial infarction and coronary artery disease. Hum. Mol. Genet. 2007, 16, 887−899. (138) Helgadottir, A.; Thorleifsson, G.; Manolescu, A.; Gretarsdottir, S.; Blondal, T.; Jonasdottir, A.; Jonasdottir, A.; Sigurdsson, A.; Baker, A.; Palsson, A.; Masson, G.; Gudbjartsson, D. F.; Magnusson, K. P.; Andersen, K.; Levey, A. I.; Backman, V. M.; Matthiasdottir, S.; Jonsdottir, T.; Palsson, S.; Einarsdottir, H.; Gunnarsdottir, S.; Gylfason, A.; Vaccarino, V.; Hooper, W. C.; Reilly, M. P.; Granger, C. B.; Austin, H.; Rader, D. J.; Shah, S. H.; Quyyumi, A. A.; Gulcher, J. R.; Thorgeirsson, G.; Thorsteinsdottir, U.; Kong, A.; Stefansson, K. A common variant on chromosome 9p21 affects the risk of myocardial infarction. Science 2007, 316, 1491−1493. (139) Kindler, J.; Bailer, U.; de Zwaan, M.; Fuchs, K.; Leisch, F.; Grun, B.; Strnad, A.; Stojanovic, M.; Windisch, J.; Lennkh-Wolfsberg, C.; El-Giamal, N.; Sieghart, W.; Kasper, S.; Aschauer, H. No association of the neuropeptide Y (Leu7Pro) and ghrelin gene (Arg51Gln, Leu72Met, Gln90Leu) single nucleotide polymorphisms with eating disorders. Nord. J. Psychiatry 2011, 65, 203−207. (140) Monteleone, P.; Tortorella, A.; Castaldo, E.; Di Filippo, C.; Maj, M. No association of the Arg51Gln and Leu72Met polymorphisms of the ghrelin gene with anorexia nervosa or bulimia nervosa. Neurosci. Lett. 2006, 398, 325−327. (141) Suchankova, P.; Jerlhag, E.; Jayaram-Lindstrom, N.; Nilsson, S.; Toren, K.; Rosengren, A.; Engel, J. A.; Franck, J. Genetic variation of the ghrelin signalling system in individuals with amphetamine dependence. PLoS One 2013, 8, e61242. (142) Landgren, S.; Jerlhag, E.; Hallman, J.; Oreland, L.; Lissner, L.; Strandhagen, E.; Thelle, D. S.; Zetterberg, H.; Blennow, K.; Engel, J. A. Genetic variation of the ghrelin signaling system in females with severe alcohol dependence. Alcohol.: Clin. Exp. Res. 2010, 34, 1519−1524. (143) Zuo, L.; Wang, K.; Zhang, X. Y.; Krystal, J. H.; Li, C. S.; Zhang, F.; Zhang, H.; Luo, X. NKAIN1-SERINC2 is a functional, replicable and genome-wide significant risk gene region specific for alcohol dependence in subjects of European descent. Drug Alcohol Depend. 2013, 129, 254−264. (144) Frank, J.; Cichon, S.; Treutlein, J.; Ridinger, M.; Mattheisen, M.; Hoffmann, P.; Herms, S.; Wodarz, N.; Soyka, M.; Zill, P.; Maier, W.; Mossner, R.; Gaebel, W.; Dahmen, N.; Scherbaum, N.; Schmal, C.; Steffens, M.; Lucae, S.; Ising, M.; Muller-Myhsok, B.; Nothen, M. M.; Mann, K.; Kiefer, F.; Rietschel, M. Genome-wide significant association between alcohol dependence and a variant in the ADH gene cluster. Addict. Biol. 2012, 17, 171−180. (145) Wade, T. D.; Gordon, S.; Medland, S.; Bulik, C. M.; Heath, A. C.; Montgomery, G. W.; Martin, N. G. Genetic variants associated with disordered eating. Int. J. Eating Disord. 2013, 46, 594−608. (146) Boraska, V.; Davis, O. S.; Cherkas, L. F.; Helder, S. G.; Harris, J.; Krug, I.; Liao, T. P.; Treasure, J.; Ntalla, I.; Karhunen, L.; KeskiRahkonen, A.; Christakopoulou, D.; Raevuori, A.; Shin, S. Y.; Dedoussis, G. V.; Kaprio, J.; Soranzo, N.; Spector, T. D.; Collier, D. A.; Zeggini, E. Genome-wide association analysis of eating disorderrelated symptoms, behaviors, and personality traits. Am. J. Med. Genet., Part B 2012, 159, 803−811. (147) Lango Allen, H.; Estrada, K.; Lettre, G.; Berndt, S. I.; Weedon, M. N.; Rivadeneira, F.; Willer, C. J.; Jackson, A. U.; Vedantam, S.; Raychaudhuri, S.; Ferreira, T.; Wood, A. R.; Weyant, R. J.; Segre, A. V.; Speliotes, E. K.; Wheeler, E.; Soranzo, N.; Park, J. H.; Yang, J.; Gudbjartsson, D.; Heard-Costa, N. L.; Randall, J. C.; Qi, L.; Vernon Smith, A.; Magi, R.; Pastinen, T.; Liang, L.; Heid, I. M.; Luan, J.; Thorleifsson, G.; Winkler, T. W.; Goddard, M. E.; Lo, K. S.; Palmer, C.; Workalemahu, T.; Aulchenko, Y. S.; Johansson, A.; Zillikens, M. C.; Feitosa, M. F.; Esko, T.; Johnson, T.; Ketkar, S.; Kraft, P.; Mangino, M.; Prokopenko, I.; Absher, D.; Albrecht, E.; Ernst, F.; Glazer, N. L.; Hayward, C.; Hottenga, J. J.; Jacobs, K. B.; Knowles, J.

W.; Kutalik, Z.; Monda, K. L.; Polasek, O.; Preuss, M.; Rayner, N. W.; Robertson, N. R.; Steinthorsdottir, V.; Tyrer, J. P.; Voight, B. F.; Wiklund, F.; Xu, J.; Zhao, J. H.; Nyholt, D. R.; Pellikka, N.; Perola, M.; Perry, J. R.; Surakka, I.; Tammesoo, M. L.; Altmaier, E. L.; Amin, N.; Aspelund, T.; Bhangale, T.; Boucher, G.; Chasman, D. I.; Chen, C.; Coin, L.; Cooper, M. N.; Dixon, A. L.; Gibson, Q.; Grundberg, E.; Hao, K.; Juhani Junttila, M.; Kaplan, L. M.; Kettunen, J.; Konig, I. R.; Kwan, T.; Lawrence, R. W.; Levinson, D. F.; Lorentzon, M.; McKnight, B.; Morris, A. P.; Muller, M.; Suh Ngwa, J.; Purcell, S.; Rafelt, S.; Salem, R. M.; Salvi, E.; Sanna, S.; Shi, J.; Sovio, U.; Thompson, J. R.; Turchin, M. C.; Vandenput, L.; Verlaan, D. J.; Vitart, V.; White, C. C.; Ziegler, A.; Almgren, P.; Balmforth, A. J.; Campbell, H.; Citterio, L.; De Grandi, A.; Dominiczak, A.; Duan, J.; Elliott, P.; Elosua, R.; Eriksson, J. G.; Freimer, N. B.; Geus, E. J.; Glorioso, N.; Haiqing, S.; Hartikainen, A. L.; Havulinna, A. S.; Hicks, A. A.; Hui, J.; Igl, W.; Illig, T.; Jula, A.; Kajantie, E.; Kilpelainen, T. O.; Koiranen, M.; Kolcic, I.; Koskinen, S.; Kovacs, P.; Laitinen, J.; Liu, J.; Lokki, M. L.; Marusic, A.; Maschio, A.; Meitinger, T.; Mulas, A.; Pare, G.; Parker, A. N.; Peden, J. F.; Petersmann, A.; Pichler, I.; Pietilainen, K. H.; Pouta, A.; Ridderstrale, M.; Rotter, J. I.; Sambrook, J. G.; Sanders, A. R.; Schmidt, C. O.; Sinisalo, J.; Smit, J. H.; Stringham, H. M.; Bragi Walters, G.; Widen, E.; Wild, S. H.; Willemsen, G.; Zagato, L.; Zgaga, L.; Zitting, P.; Alavere, H.; Farrall, M.; McArdle, W. L.; Nelis, M.; Peters, M. J.; Ripatti, S.; van Meurs, J. B.; Aben, K. K.; Ardlie, K. G.; Beckmann, J. S.; Beilby, J. P.; Bergman, R. N.; Bergmann, S.; Collins, F. S.; Cusi, D.; den Heijer, M.; Eiriksdottir, G.; Gejman, P. V.; Hall, A. S.; Hamsten, A.; Huikuri, H. V.; Iribarren, C.; Kahonen, M.; Kaprio, J.; Kathiresan, S.; Kiemeney, L.; Kocher, T.; Launer, L. J.; Lehtimaki, T.; Melander, O.; Mosley, T. H., Jr.; Musk, A. W.; Nieminen, M. S.; O’Donnell, C. J.; Ohlsson, C.; Oostra, B.; Palmer, L. J.; Raitakari, O.; Ridker, P. M.; Rioux, J. D.; Rissanen, A.; Rivolta, C.; Schunkert, H.; Shuldiner, A. R.; Siscovick, D. S.; Stumvoll, M.; Tonjes, A.; Tuomilehto, J.; van Ommen, G. J.; Viikari, J.; Heath, A. C.; Martin, N. G.; Montgomery, G. W.; Province, M. A.; Kayser, M.; Arnold, A. M.; Atwood, L. D.; Boerwinkle, E.; Chanock, S. J.; Deloukas, P.; Gieger, C.; Gronberg, H.; Hall, P.; Hattersley, A. T.; Hengstenberg, C.; Hoffman, W.; Lathrop, G. M.; Salomaa, V.; Schreiber, S.; Uda, M.; Waterworth, D.; Wright, A. F.; Assimes, T. L.; Barroso, I.; Hofman, A.; Mohlke, K. L.; Boomsma, D. I.; Caulfield, M. J.; Cupples, L. A.; Erdmann, J.; Fox, C. S.; Gudnason, V.; Gyllensten, U.; Harris, T. B.; Hayes, R. B.; Jarvelin, M. R.; Mooser, V.; Munroe, P. B.; Ouwehand, W. H.; Penninx, B. W.; Pramstaller, P. P.; Quertermous, T.; Rudan, I.; Samani, N. J.; Spector, T. D.; Volzke, H.; Watkins, H.; Wilson, J. F.; Groop, L. C.; Haritunians, T.; Hu, F. B.; Kaplan, R. C.; Metspalu, A.; North, K. E.; Schlessinger, D.; Wareham, N. J.; Hunter, D. J.; O’Connell, J. R.; Strachan, D. P.; Wichmann, H. E.; Borecki, I. B.; van Duijn, C. M.; Schadt, E. E.; Thorsteinsdottir, U.; Peltonen, L.; Uitterlinden, A. G.; Visscher, P. M.; Chatterjee, N.; Loos, R. J.; Boehnke, M.; McCarthy, M. I.; Ingelsson, E.; Lindgren, C. M.; Abecasis, G. R.; Stefansson, K.; Frayling, T. M.; Hirschhorn, J. N. Hundreds of variants clustered in genomic loci and biological pathways affect human height. Nature 2010, 467, 832−838. (148) Gueorguiev, M.; Lecoeur, C.; Benzinou, M.; Mein, C. A.; Meyre, D.; Vatin, V.; Weill, J.; Heude, B.; Grossman, A. B.; Froguel, P.; Korbonits, M. A genetic study of the ghrelin and growth hormone secretagogue receptor (GHSR) genes and stature. Ann. Hum. Genet. 2009, 73, 1−9. (149) Inoue, H.; Kangawa, N.; Kinouchi, A.; Sakamoto, Y.; Kimura, C.; Horikawa, R.; Shigematsu, Y.; Itakura, M.; Ogata, T.; Fujieda, K.; Japan Growth Genome Consortium.. Identification and functional analysis of novel human growth hormone secretagogue receptor (GHSR) gene mutations in Japanese subjects with short stature. J. Clin. Endocrinol. Metab. 2011, 96, E373−E378. (150) Muller, T. D.; Tschop, M. H.; Jarick, I.; Ehrlich, S.; Scherag, S.; Herpertz-Dahlmann, B.; Zipfel, S.; Herzog, W.; de Zwaan, M.; Burghardt, R.; Fleischhaker, C.; Klampfl, K.; Wewetzer, C.; Herpertz, S.; Zeeck, A.; Tagay, S.; Burgmer, M.; Pfluger, P. T.; Scherag, A.; Hebebrand, J.; Hinney, A. Genetic variation of the ghrelin activator S

dx.doi.org/10.1021/jm5003183 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Perspective

gene ghrelin O-acyltransferase (GOAT) is associated with anorexia nervosa. J. Psychiatr. Res. 2011, 45, 706−711. (151) Costantino, L.; Barlocco, D. Ghrelin receptor modulators and their therapeutic potential. Future Med. Chem. 2009, 1, 157−177. (152) Carpino, P. A.; Ho, G. Modulators of the ghrelin system as potential treatments for obesity and diabetes. Expert Opin. Ther. Pat. 2008, 18, 1253−1263. (153) Morphy, R. The influence of target family and functional activity on the physicochemical properties of pre-clinical compounds. J. Med. Chem. 2006, 49, 2969−2978. (154) Leeson, P. D.; Springthorpe, B. The influence of drug-like concepts on decision-making in medicinal chemistry. Nat. Rev. Drug Discovery 2007, 6, 881−890. (155) Hitchcock, S. A. Structural modifications that alter the Pglycoprotein efflux properties of compounds. J. Med. Chem. 2012, 55, 4877−4895. (156) Gupta, R. R.; Gifford, E. M.; Liston, T.; Waller, C. L.; Hohman, M.; Bunin, B. A.; Ekins, S. Using open source computational tools for predicting human metabolic stability and additional absorption, distribution, metabolism, excretion, and toxicity properties. Drug Metab. Dispos. 2010, 38, 2083−2090. (157) Serby, M. D.; Zhao, H.; Szczepankiewicz, B. G.; Kosogof, C.; Xin, Z.; Liu, B.; Liu, M.; Nelson, L. T.; Kaszubska, W.; Falls, H. D.; Schaefer, V.; Bush, E. N.; Shapiro, R.; Droz, B. A.; Knourek-Segel, V. E.; Fey, T. A.; Brune, M. E.; Beno, D. W.; Turner, T. M.; Collins, C. A.; Jacobson, P. B.; Sham, H. L.; Liu, G. 2,4-Diaminopyrimidine derivatives as potent growth hormone secretagogue receptor antagonists. J. Med. Chem. 2006, 49, 2568−2578. (158) Liu, B.; Liu, G.; Xin, Z.; Serby, M. D.; Zhao, H.; Schaefer, V. G.; Falls, H. D.; Kaszubska, W.; Collins, C. A.; Sham, H. L. Novel isoxazole carboxamides as growth hormone secretagogue receptor (GHS-R) antagonists. Bioorg. Med. Chem. Lett. 2004, 14, 5223−5226. (159) Zhao, H.; Xin, Z.; Liu, G.; Schaefer, V. G.; Falls, H. D.; Kaszubska, W.; Collins, C. A.; Sham, H. L. Discovery of tetralin carboxamide growth hormone secretagogue receptor antagonists via scaffold manipulation. J. Med. Chem. 2004, 47, 6655−6657. (160) Rudolph, J.; Esler, W. P.; O’Connor, S.; Coish, P. D.; Wickens, P. L.; Brands, M.; Bierer, D. E.; Bloomquist, B. T.; Bondar, G.; Chen, L.; Chuang, C. Y.; Claus, T. H.; Fathi, Z.; Fu, W.; Khire, U. R.; Kristie, J. A.; Liu, X. G.; Lowe, D. B.; McClure, A. C.; Michels, M.; Ortiz, A. A.; Ramsden, P. D.; Schoenleber, R. W.; Shelekhin, T. E.; Vakalopoulos, A.; Tang, W.; Wang, L.; Yi, L.; Gardell, S. J.; Livingston, J. N.; Sweet, L. J.; Bullock, W. H. Quinazolinone derivatives as orally available ghrelin receptor antagonists for the treatment of diabetes and obesity. J. Med. Chem. 2007, 50, 5202−5216. (161) Moulin, A.; Demange, L.; Ryan, J.; Mousseaux, D.; Sanchez, P.; Berge, G.; Gagne, D.; Perrissoud, D.; Locatelli, V.; Torsello, A.; Galleyrand, J.-C.; Fehrentz, J.-A.; Martinez, J. New trisubstituted 1,2,4triazole derivatives as potent ghrelin receptor antagonists. Synthesis and pharmacological in vitro and in vivo evaluations. J. Med. Chem. 2008, 51, 689−693. (162) Ge, M.; Cline, E.; Yang, L.; Mills, S. G. Preparation of substituted pyrazoles as ghrelin receptor antagonists. WO2008008286A2, 2008. (163) Ertl, P.; Rohde, B.; Selzer, P. Fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties. J. Med. Chem. 2000, 43, 3714−3717. (164) A combination of keywords and index terms were used to retrieve patent data from Thomson Reuters Cortellis (Thomson Reuters, London and Philadelphia, 2014, available from http:// thomsonreuters.com/cortellis-competitive-intelligence/), Thomson Reuters Integrity (Thomson Reuters, Barcelona, London, Philadelphia, 2014; available from http://thomsonreuters.com/integrity/), and SciFinder. Patents unique to each of these databases were further evaluated for relevancy. However, patents found in multiple databases were used without manual evaluation. Finally, patent numbers from all three resources were pooled in PatBase (owned by RWS Information

Ltd. and Minesoft Ltd.), where the duplicate families were identified and removed. (165) Pasternak, A.; Goble, S. D.; deJesus, R. K.; Hreniuk, D. L.; Chung, C. C.; Tota, M. R.; Mazur, P.; Feighner, S. D.; Howard, A. D.; Mills, S. G.; Yang, L. Discovery and optimization of novel 4[(aminocarbonyl)amino]-N-[4-(2-aminoethyl)phenyl]benzenesulfonamide ghrelin receptor antagonists. Bioorg. Med. Chem. Lett. 2009, 19, 6237−6240. (166) Longo, K. A.; Govek, E. K.; Nolan, A.; McDonagh, T.; Charoenthongtrakul, S.; Giuliana, D. J.; Morgan, K.; Hixon, J.; Zhou, C.; Kelder, B.; Kopchick, J. J.; Saunders, J. O.; Navia, M. A.; Curtis, R.; DiStefano, P. S.; Geddes, B. J. Pharmacologic inhibition of ghrelin receptor signaling is insulin sparing and promotes insulin sensitivity. J. Pharmacol. Exp. Ther. 2011, 339, 115−124. (167) Nolan, A. L. Compositions and methods for preserving pancreatic islet mass. WO2010022213A2, 2010. (168) Elixir Pharmaceuticals Signs Option Agreement with a Major Pharmaceutical Company; Completes $12M Equity Financing. http:// www.businesswire.com/news/home/20090519005994/en/ElixirPharmaceuticals-Signs-Option-Agreement-Major-Pharmaceutical. (169) Yu, M.; Lizarzaburu, M.; Beckmann, H.; Connors, R.; Dai, K.; Haller, K.; Li, C.; Liang, L.; Lindstrom, M.; Ma, J.; Motani, A.; Wanska, M.; Zhang, A.; Li, L.; Medina, J. C. Identification of piperazinebisamide GHSR antagonists for the treatment of obesity. Bioorg. Med. Chem. Lett. 2010, 20, 1758−1762. (170) Mihalic, J. T.; Kim, Y. J.; Lizarzaburu, M.; Chen, X.; Deignan, J.; Wanska, M.; Yu, M.; Fu, J.; Chen, X.; Zhang, A.; Connors, R.; Liang, L.; Lindstrom, M.; Ma, J.; Tang, L.; Dai, K.; Li, L. Discovery of a new class of ghrelin receptor antagonists. Bioorg. Med. Chem. Lett. 2012, 22, 2046−2051. (171) Sabbatini, F. M.; Melotto, S.; Bernasconi, G.; Bromidge, S. M.; D’Adamo, L.; Rinaldi, M.; Savoia, C.; Mundi, C.; Di Francesco, C.; Zonzini, L.; Costantini, V. J.; Perini, B.; Valerio, E.; Pozzan, A.; Perdona, E.; Visentini, F.; Corsi, M.; Di Fabio, R. Azabicyclo[3.1.0]hexane-1-carbohydrazides as potent and selective GHSR1a ligands presenting a specific in vivo behavior. ChemMedChem 2011, 6, 1981− 1985. (172) Perdona, E.; Faggioni, F.; Buson, A.; Sabbatini, F. M.; Corti, C.; Corsi, M. Pharmacological characterization of the ghrelin receptor antagonist, GSK1614343 in rat RC-4B/C cells natively expressing GHS type 1a receptors. Eur. J. Pharmacol. 2011, 650, 178−183. (173) Costantini, V. J.; Vicentini, E.; Sabbatini, F. M.; Valerio, E.; Lepore, S.; Tessari, M.; Sartori, M.; Michielin, F.; Melotto, S.; Bifone, A.; Pich, E. M.; Corsi, M. GSK1614343, a novel ghrelin receptor antagonist, produces an unexpected increase of food intake and body weight in rodents and dogs. Neuroendocrinology 2011, 94, 158−168. (174) Holst, B.; Mokrosinski, J.; Lang, M.; Brandt, E.; Nygaard, R.; Frimurer, T. M.; Beck-Sickinger, A. G.; Schwartz, T. W. Identification of an efficacy switch region in the ghrelin receptor responsible for interchange between agonism and inverse agonism. J. Biol. Chem. 2007, 282, 15799−15811. (175) Holst, B.; Lang, M.; Brandt, E.; Bach, A.; Howard, A.; Frimurer, T. M.; Beck-Sickinger, A.; Schwartz, T. W. Ghrelin receptor inverse agonists: identification of an active peptide core and its interaction epitopes on the receptor. Mol. Pharmacol. 2006, 70, 936− 946. (176) Hoveyda, H. R.; Marsault, E.; Gagnon, R.; Mathieu, A. P.; Vezina, M.; Landry, A.; Wang, Z.; Benakli, K.; Beaubien, S.; SaintLouis, C.; Brassard, M.; Pinault, J. F.; Ouellet, L.; Bhat, S.; Ramaseshan, M.; Peng, X.; Foucher, L.; Beauchemin, S.; Bherer, P.; Veber, D. F.; Peterson, M. L.; Fraser, G. L. Optimization of the potency and pharmacokinetic properties of a macrocyclic ghrelin receptor agonist (Part I): development of ulimorelin (TZP-101) from hit to clinic. J. Med. Chem. 2011, 54, 8305−8320. (177) Hoveyda, H.; Marsault, E.; Thomas, H.; Fraser, G.; Beaubien, S.; Mathieu, A.; Beignet, J.; Bonin, M.-A.; Phoenix, S.; Drutz, D.; Peterson, M.; Beauchemin, S.; Brassard, M.; Vezina, M. Preparation of macrocyclic ghrelin receptor antagonists and inverse agonists. WO2011053821A1, 2011. T

dx.doi.org/10.1021/jm5003183 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Perspective

(178) Le Poul, E.; Hisada, S.; Mizuguchi, Y.; Dupriez, V. J.; Burgeon, E.; Detheux, M. Adaptation of aequorin functional assay to high throughput screening. J. Biomol. Screening 2002, 7, 57−65. (179) Bergsdorf, C.; Kropp-Goerkis, C.; Kaehler, I.; Ketscher, L.; Boemer, U.; Parczyk, K.; Bader, B. A one-day, dispense-only IP-One HTRF assay for high-throughput screening of Galphaq proteincoupled receptors: towards cells as reagents. Assay Drug Dev. Technol. 2008, 6, 39−53. (180) Hanrahan, P.; Bell, J.; Bottomley, G.; Bradley, S.; Clarke, P.; Curtis, E.; Davis, S.; Dawson, G.; Horswill, J.; Keily, J.; Moore, G.; Rasamison, C.; Bloxham, J. Substituted azaquinazolinones as modulators of GHSr-1a for the treatment of type II diabetes and obesity. Bioorg. Med. Chem. Lett. 2012, 22, 2271−2278. (181) Bloxham, J.; Bradley, S. E.; Sambrook-Smith, C. P.; Smyth, D.; Keily, J.; Dawson, G. J.; Rasamison, C. M.; Bell, J. C. Preparation of piperidine amides as modulators of ghrelin receptor. WO2011117254A1, 2011. (182) Garcia, R. S.; Pietra, C.; Giuliano, C.; Li, Z. Asymmetric ureas as ghrelin receptor modulators. WO2012116176A2, 2012. (183) Puleo, L.; Marini, P.; Avallone, R.; Zanchet, M.; Bandiera, S.; Baroni, M.; Croci, T. Synthesis and pharmacological evaluation of indolinone derivatives as novel ghrelin receptor antagonists. Bioorg. Med. Chem. 2012, 20, 5623−5236. (184) Tokunaga, T.; Hume, W. E.; Umezome, T.; Okazaki, K.; Ueki, Y.; Kumagai, K.; Hourai, S.; Nagamine, J.; Seki, H.; Taiji, M.; Noguchi, H.; Nagata, R. Oxindole derivatives as orally active potent growth hormone secretagogues. J. Med. Chem. 2001, 44, 4641−4649. (185) McCoull, W.; Barton, P.; Broo, A.; Brown, A. J. H.; Clarke, D. S.; Coope, G.; Davies, R. D. M.; Dossetter, A. G.; Kelly, E. E.; Knerr, L.; MacFaul, P.; Holmes, J. L.; Martin, N.; Moore, J. E.; Morgan, D.; Newton, C.; Osterlund, K.; Robb, G. R.; Rosevere, E.; Selmi, N.; Stokes, S.; Svensson, T. S.; Ullah, V. B. K.; Williams, E. J. Identification of pyrazolo-pyrimidinones as GHS-R1a antagonists and inverse agonists for the treatment of obesity. Med. Chem. Commun. 2013, 4, 456−462. (186) Rudolph, J.; Esler, W. P.; O’Connor, S.; Coish, P. D.; Wickens, P. L.; Brands, M.; Bierer, D. E.; Bloomquist, B. T.; Bondar, G.; Chen, L.; Chuang, C. Y.; Claus, T. H.; Fathi, Z.; Fu, W.; Khire, U. R.; Kristie, J. A.; Liu, X. G.; Lowe, D. B.; McClure, A. C.; Michels, M.; Ortiz, A. A.; Ramsden, P. D.; Schoenleber, R. W.; Shelekhin, T. E.; Vakalopoulos, A.; Tang, W.; Wang, L.; Yi, L.; Gardell, S. J.; Livingston, J. N.; Sweet, L. J.; Bullock, W. H. Quinazolinone derivatives as orally available ghrelin receptor antagonists for the treatment of diabetes and obesity. J. Med. Chem. 2007, 50, 5202−5216. (187) Edwards, M. P.; Price, D. A. Role of physicochemical properties and ligand lipophilicity efficiency in addressing drug safety risks. In Annual Reports in Medicinal Chemistry; John, E. M., Ed.; Academic Press: San Diego, CA, 2010; Vol. 45, Chapter 23, pp 380− 391. (188) Kung, D. W.; Coffey, S. B.; Jones, R. M.; Cabral, S.; Jiao, W.; Fichtner, M.; Carpino, P. A.; Rose, C. R.; Hank, R. F.; Lopaze, M. G.; Swartz, R.; Chen, H. T.; Hendsch, Z.; Posner, B.; Wielis, C. F.; Manning, B.; Dubins, J.; Stock, I. A.; Varma, S.; Campbell, M.; DeBartola, D.; Kosa-Maines, R.; Steyn, S. J.; McClure, K. F. Identification of spirocyclic piperidine-azetidine inverse agonists of the ghrelin receptor. Bioorg. Med. Chem. Lett. 2012, 22, 4281−4287. (189) McClure, K. F.; Jackson, M.; Cameron, K. O.; Kung, D. W.; Perry, D. A.; Orr, S. T.; Zhang, Y.; Kohrt, J.; Tu, M.; Gao, H.; Fernando, D.; Jones, R.; Erasga, N.; Wang, G.; Polivkova, J.; Jiao, W.; Swartz, R.; Ueno, H.; Bhattacharya, S. K.; Stock, I. A.; Varma, S.; Bagdasarian, V.; Perez, S.; Kelly-Sullivan, D.; Wang, R.; Kong, J.; Cornelius, P.; Michael, L.; Lee, E.; Janssen, A.; Steyn, S. J.; Lapham, K.; Goosen, T. Identification of potent, selective, CNS-targeted inverse agonists of the ghrelin receptor. Bioorg. Med. Chem. Lett. 2013, 23, 5410−5414. (190) Bhattacharya, S. K.; Andrews, K.; Beveridge, R.; Cameron, K. O.; Chen, C.; Dunn, M.; Fernando, D.; Gao, H.; Hepworth, D.; Jackson, V. M.; Khot, V.; Kong, J.; Kosa, R. E.; Lapham, K.; Loria, P. M.; Londregan, A. T.; McClure, K. F.; Orr, S. T. M.; Patel, J.; Rose, C.;

Saenz, J.; Stock, I. A.; Storer, G.; VanVolkenburg, M.; Vrieze, D.; Wang, G.; Xiao, J.; Zhang, Y. Discovery of PF-5190457, a potent, selective, and orally bioavailable ghrelin receptor inverse agonist clinical candidate. ACS Med. Chem. Lett. 2014, 5, 474−479. (191) Compound 22b, also known as PF-04628935, is now commercially available from Sigma Aldrich (catalog no. PZ0228). (192) Bagal, S. K.; Bungay, P. J. Minimizing drug exposure in the CNS while maintaining good oral absorption. ACS Med. Chem. Lett. 2012, 3, 948−950.

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